Human anti-pd-1, pd-l1, and pd-l2 antibodies and uses therefor

ABSTRACT

The present invention is based, in part, on the identification of novel human anti-PD-1, PD-L1, and PD-L2 antibodies. Accordingly, the invention relates to compositions and methods for diagnosing, prognosing, and treating conditions that would benefit from modulating PD-1, PD-L1, and/or PD-L2 activity (e.g., persistent infectious diseases, autoimmune diseases, asthma, transplant rejection, inflammatory disorders and tumors) using the novel human anti-PD-1, PD-L1, and PD-L2 antibodies described herein.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/802,172, filed Sep. 25, 2009 which is a continuation of U.S.application Ser. No. 13/120,406, filed Sep. 25, 2009, now U.S. Pat. No.8,552,154, which is a National Phase application under 35 U.S.C. §371 ofInternational Application No. PCT/US2009/058475, filed on Sep. 25, 2009,which claims priority benefit of U.S. Provisional Application No.61/100,534, filed Sep. 26, 2008, all of which are hereby incorporated byreference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 677192000102SeqList.TXT,date recorded: Oct. 1, 2015, size: 71 KB).

BACKGROUND OF THE INVENTION

For T cells to respond to foreign polypeptides, at least two signalsmust be provided by antigen-presenting cells (APCs) to resting Tlymphocytes (Jenkins, M. and Schwartz, R. (1987) J. Exp. Med.165:302-319; Mueller, D. L. et al. (1990) J. Immunol. 144:3701-3709).The first signal, which confers specificity to the immune response, istransduced via the T cell receptor (TCR) following recognition offoreign antigenic peptide presented in the context of the majorhistocompatibility complex (MHC). The second signal, termedcostimulation, induces T cells to proliferate and become functional(Lenschow et al. (1996) Annu. Rev. Immunol. 14:233). Costimulation isneither antigen-specific, nor MHC-restricted, and is provided bydistinct cell surface molecules expressed by APCs (Jenkins, M. K. et al.(1988) J. Immunol. 140:3324-3330; Linsley, P. S. et al. (1991) J. Exp.Med 173:721-730; Gimmi, C. D. et al. (1991) Proc. Natl. Acad Sci. USA88:6575-6579; Young, J. W. et al. (1992) J. Clin. Invest. 90:229-237;Koulova, L. et al. (1991) J. Exp. Med. 173:759-762; Reiser, H. et al.(1992) Proc. Natl. Acad Sci. USA 89:271-275; van-Seventer, G. A. et al.(1990) J. Immunol. 144:4579-4586; LaSalle, J. M. et al. (1991) J.Immunol. 147:774-80; Dustin, M. I. et al. (1989) J. Exp. Med. 169:503;Armitage, R. J. et al. (1992) Nature 357:80-82; Liu, Y. et al. (1992) J.Exp. Med 175:437-445).

The proteins B7-1 (CD80) and B7-2 (CD86) are critical costimulatorymolecules (Freeman et al. (1991) J. Exp. Med. 174:625; Freeman et al.(1989) J. Immunol. 143:2714; Azuma et al. (1993) Nature 366:76; Freemanet al. (1993) Science 262:909). B7-2 plays a predominant role duringprimary immune responses, while B7-1, which is upregulated later duringan immune response, may be important for prolonging primary T cellresponses or costimulating secondary T cell responses (Bluestone (1995)Immunity 2:555).

CD28 is a ligand for both B7-1 and B7-2 that is constitutively expressedby resting T cells and increases in expression following T cellactivation. Ligation of CD28 in conjunction with a TCR signal results intransduction of a costimulatory signal that induces T cells toproliferate and secrete IL-2 (Linsley, P. S. et al. (1991) J. Exp. Med.173:721-730; Gimmi, C. D. et al. (1991) Proc. Natl. Acad. Sci. USA88:6575-6579; June, C. H. et al. (1990) Immunol. Today 11:211-6;Harding, F. A. et al. (1992) Nature 356:607-609). A second B7-1 and B7-2ligand, CTLA4 (CD152), is homologous to CD28 but not expressed byresting T cells. CTLA4 expression occurs following T cell activation(Brunet, J. F. et al. (1987) Nature 328:267-270). Ligation of CTLA4results in transduction of an inhibitory signal that prevents T cellproliferation and cytokine secretion. Thus, CTLA4 is a critical negativeregulator of T cell responses (Waterhouse et al. (1995) Science 270:985)(Allison and Krummel (1995) Science 270:932). The third member of theCD28 family to be discovered is ICOS (Hutloff et al. (1999) Nature397:263; WO 98/38216). Ligation of ICOS by its ligand (ICOS-L) resultsin high levels of cytokine expression, but limited T cell expansion(Riley J. L. et al. (2001) J. Immunol. 166:4943-48; Aicher A. et al.(2000) J. Immunol. 164:4689-96; Mages H. W. et al. (2000) Eur. JImmunol. 30:1040-7; Brodie D. et al. (2000) Curr. Biol. 10:333-6; LingV. et al. (2000) J. Immunol. 164:1653-7; Yoshinaga S. K. et al. (1999)Nature 402:827-32). If T cells are stimulated through the T cellreceptor in the absence of a costimulatory signal, they becomenonresponsive, anergic, or die.

The importance of the B7:CD28/CTLA4/ICOS costimulatory pathway has beendemonstrated in vitro and in several in vivo model systems. Blockade ofthis costimulatory pathway results in the development of antigenspecific tolerance in murine and human systems (Harding, F. A. et al.(1992) Nature 356:607 609; Lenschow, D. J. et al. (1992) Science 257:789792; Turka, L. A. et al. (1992) Proc. Natl. Acad. Sci. USA 89:1110211105; Gimmi, C. D. et al. (1993) Proc. Natl. Acad. Sci. USA 90:65866590; Boussiotis, V. et al. (1993) J. Exp. Med. 178:1753 1763).Conversely, expression of B7 by B7-negative murine tumor cells inducesT-cell mediated specific immunity accompanied by tumor rejection andlong lasting protection to tumor challenge (Chen, L. et al. (1992) Cell71:1093 1102; Townsend, S. E. and Allison, J. P. (1993) Science 259:368370; Baskar, S. et al. (1993) Proc. Natl. Acad. Sci. 90:5687 5690.).Therefore, manipulation of the costimulatory pathways offers greatpotential to stimulate or suppress immune responses in humans.

The discovery of more members of the B7-1 and CD28 families has revealedadditional pathways that provide costimulatory and inhibitory secondsignals to T cells. One of the newer pathways is represented by theprogrammed death 1 (PD-1; also known as CD279) receptor and its ligands,PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273). PD-1 is a member of theCD28/CTLA4 family that is expressed on activated, but not resting Tcells (Nishimura et al. (1996) Int. Immunol. 8:773). Ligation of PD-1 byits ligands mediates an inhibitory signal that results in reducedcytokine production, and reduced T cell survival (Nishimura et al.(1999) Immunity 11:141; Nishimura et al. (2001) Science 291:319;Chemnitz et al. (2004) J. Immunol. 173:945).

PD-L1 is a B7 family member that is expressed on many cell types,including APCs and activated T cells (Yamazaki et al. (2002) J. Immunol.169:5538). PD-L1 binds to both PD-1 and B7-1. Both binding ofT-cell-expressed B7-1 by PD-L1 and binding of T-cell-expressed PD-L1 byB7-1 result in T cell inhibition (Butte et al. (2007) Immunity 27:111).There is also evidence that, like other B7 family members, PD-L1 canalso provide costimulatory signals to T cells (Subudhi et al. (2004) J.Clin. Invest. 113:694; Tamura et al. (2001) Blood 97:1809).

PD-L2 is a B7 family member expressed on various APCs, includingdendritic cells, macrophages and bone-marrow derived mast cells (Zhonget al. (2007) Eur. J. Immunol. 37:2405). APC-expressed PD-L2 is able toboth inhibit T cell activation through ligation of PD-1 and costimulateT cell activation, through a PD-1 independent mechanism (Shin et al.(2005) J. Exp. Med. 201:1531). In addition, ligation of dendriticcell-expressed PD-L2 results in enhanced dendritic cell cytokineexpression and survival (Radhakrishnan et al. (2003) J. Immunol.37:1827; Nguyen et al. (2002) J. Exp. Med. 196:1393). The structure andexpression of PD-1, PD-L1, and PD-L2, as well as signalingcharacteristics and functions of these molecules in the context ofregulating T cell activation and tolerance (e.g., therapeutic effects)are reviewed in greater detail in Kier et al. (2008) Ann. Rev. Immunol.26:677, which is herein incorporated by reference in its entirety.Manipulation of this and other costimulatory pathways offers greatpotential to stimulate or suppress immune responses in humans and a needexists for compositions and methods useful for effecting suchmanipulations.

SUMMARY OF THE INVENTION

The present invention is based on the generation and isolation of novelcomposite, human monoclonal antibodies which specifically bind to humanPD-1, human PD-L1, and human PD-L2, as well as the characterization ofsuch novel antibodies and the demonstration of their therapeutic valuein treating a variety of conditions mediated by PD-1, PD-L1, and/orPD-L2. Common techniques used to humanize murine antibodies frequentlyproduce humanized antibodies that have reduced antigen bindingaffinities compared to the original murine antibodies (Almagro andFransson (2008) Frontiers in Bioscience 13:1619-1633; Foote and Winter(1992) J. Mol. Biol. 224:487-499; Hwang et al. (2005) Methods 36:35-42). Surprisingly, the composite, human antibodies of the presentinvention have been shown to bind to PD-1, PD-L1 or PD-L2 withaffinities closely approximating those of the murine antibodies.Furthermore, conventional humanization techniques produce humanizedantibodies that retain some murine sequence. As a result, suchantibodies can retain immunogenicity when administered to humans. Forexample, the humanized antibody CAMPATH® elicits immunogenicity in about50% of patients. The composite, human antibodies of the presentinvention, on the other hand, are completely derived from sequences ofhuman origin. Therefore, they are likely to be significantly lessimmunogenic and more therapeutically effective and useful whenadministered to human patients than other anti-human PD-1, PD-L1, and/orPD-L2 antibodies. Accordingly, the composite, human antibodies of thepresent invention provide an improved means for treating and preventingdisorders mediated by PD-1, PD-L1, and/or PD-L2, attributable in part totheir unique specificity, affinity, structure, functional activity andthe fact that they are derived from human antibody sequences. Thepresent invention is also based on the discovery of new therapeuticapplications, including treatment of persistent infectious diseases,asthma, inflammatory diseases, and cancers, by administering thecomposite, human antibodies described herein.

One embodiment of the invention is an isolated antibody, or anantigen-binding fragment thereof, that binds to a PD-1 protein, a PD-L1protein, or a PD-L2 protein (such as human PD-1, PD-L1, or PD-L2protein), wherein the isolated antibody, or antigen-binding fragmentthereof, is chimeric, humanized, composite, human or human, andcomprising one, two, three, four, five, or six CDR sequences selectedfrom the group consisting of SEQ ID NO: 7-24.

The invention also provides an isolated antibody, or an antigen-bindingfragment thereof, that binds to a PD-1 protein (such as a PD-1 proteincomprising the amino acid sequence of SEQ ID NO:2), wherein the isolatedantibody, or antigen-binding fragment thereof, is chimeric, humanized,composite, human or human, and comprising a heavy chain variable regionsequence comprising SEQ ID NOs:7-9 (CDR1 sequence of SEQ ID NO:7, CDR2sequence of SEQ ID NO:8, and CDR3 sequence of SEQ ID NO:9) and/or alight chain variable region sequence comprising SEQ ID NO:10-12 (CDR1sequence of SEQ ID NO:10, CDR2 sequence of SEQ ID NO:11, and CDR3sequence of SEQ ID NO:12).

The invention also provides an isolated antibody, or an antigen-bindingfragment thereof, that binds to a PD-L1 protein (such as a PD-L1 proteincomprising the amino acid sequence of SEQ ID NO:4), wherein the isolatedantibody, or antigen-binding fragment thereof, is chimeric, humanized,composite, human or human, and comprising a heavy chain variable regionsequence comprising SEQ ID NOs:13-15 (CDR1 sequence of SEQ ID NO:13,CDR2 sequence of SEQ ID NO:14, and CDR3 sequence of SEQ ID NO:15),and/or a light chain variable region sequence comprising SEQ ID NO:16-18(CDR1 sequence of SEQ ID NO:16, CDR2 sequence of SEQ ID NO:17, and CDR3sequence of SEQ ID NO:18).

The invention also provides an isolated antibody, or an antigen-bindingfragment thereof, that binds to a PD-L2 protein (such as a PD-L2 proteincomprising the amino acid sequence of SEQ ID NO:6), wherein the isolatedantibody, or antigen-binding fragment thereof, is chimeric, humanized,composite, human or human, and comprising a heavy chain variable regionsequence comprising SEQ ID NOs:19-21 (CDR1 sequence of SEQ ID NO:19,CDR2 sequence of SEQ ID NO:20, and CDR3 sequence of SEQ ID NO:21),and/or a light chain variable region sequence comprising SEQ ID NO:22-24(CDR1 sequence of SEQ ID NO:22, CDR2 sequence of SEQ ID NO:23, and CDR3sequence of SEQ ID NO:24).

The invention also includes an isolated antibody, or an antigen-bindingfragment thereof, that binds to a PD-1 protein, a PD-L1 protein, or aPD-L2 protein (such as human PD-1, PD-L1, or PD-L2 protein) wherein theisolated antibody, or antigen-binding fragment thereof, is chimeric,humanized, composite, and/or human, and comprising a heavy chainsequence selected from the group consisting of SEQ ID NO: 25-29, 34-38,or 43-47 or a sequence with at least about 95%, 96%, 97%, 98%, 99%,99.5%, 99.9% or more identical homology to SEQ ID NO: 25-29, 34-38, or43-47, and/or a light chain sequence selected from the group consistingof SEQ ID NO: 30-33, 39-42, or 48-51, or a sequence with at least about95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more homology to SEQ ID NO:30-33, 39-42, or 48-51.

The invention also provides an isolated antibody, or an antigen-bindingfragment thereof, that binds to a PD-1 protein comprising the amino acidsequence of SEQ ID NO:2, wherein the isolated antibody, orantigen-binding fragment thereof, is chimeric, humanized, composite, orhuman, and comprising a heavy chain sequence selected from the groupconsisting of SEQ ID NO: 25-29, or a sequence with at least about 95%,96%, 97%, 98%, 99%, 99.5%, 99.9% or more identical or homology to SEQ IDNO: 25-29, and/or a light chain sequence selected from the groupconsisting of SEQ ID NO: 30-33, or a sequence with at least about 95%,96%, 97%, 98%, 99%, 99.5%, 99.9% or more identical or homology to SEQ IDNO: 30-33. For example, the antibody or antigen binding fragment thereofcomprises a heavy chain variable region sequence of SEQ ID NO: 27 or 28,and a light chain variable region sequence of SEQ ID NOs: 32 or 33. Insome embodiments, the antibody or antigen binding fragment thereofcomprises a heavy chain variable region sequence of SEQ ID NO: 28, and alight chain variable region sequence of SEQ ID NOs: 32.

The invention also provides an isolated antibody, or an antigen-bindingfragment thereof, that binds to a PD-L1 protein comprising the aminoacid sequence of SEQ ID NO:4, wherein the isolated antibody, orantigen-binding fragment thereof, is chimeric, humanized, composite, orhuman, and comprising a heavy chain sequence selected from the groupconsisting of SEQ ID NO: 34-38, or a sequence with at least about 95%,96%, 97%, 98%, 99%, 99.5%, 99.9% or more identical or homology to SEQ IDNO: 34-38, and/or a light chain sequence selected from the groupconsisting of SEQ ID NO: 39-42, or a sequence with at least about 95%,96%, 97%, 98%, 99%, 99.5%, 99.9% or more identical or homology to SEQ IDNO: 39-42. For example, the antibody or antigen binding fragment thereofcomprises a heavy chain variable region sequence of SEQ ID NO: 35 or 37,and a light chain variable region sequence of SEQ ID NO: 39, 40 or 42.In some embodiments, the antibody or antigen binding fragment thereofcomprises a heavy chain variable region sequence of SEQ ID NO: 35, and alight chain variable region sequence of SEQ ID NO: 42.

The invention also provides an isolated antibody, or an antigen-bindingfragment thereof, that binds to a PD-L2 protein comprising the aminoacid sequence of SEQ ID NO:6, wherein the isolated antibody, orantigen-binding fragment thereof, is chimeric, humanized, composite, orhuman, and comprising a heavy chain sequence selected from the groupconsisting of SEQ ID NO: 43-47, or a sequence with at least about 95%,96%, 97%, 98%, 99%, 99.5%, 99.9% or more identical or homology to SEQ IDNO: 43-47, and/or a light chain sequence selected from the groupconsisting of SEQ ID NO: 48-51, or a sequence with at least about 95%,96%, 97%, 98%, 99%, 99.5%, 99.9% or more identical or homology to SEQ IDNO: 48-51. For example, the antibody or antigen binding fragment thereofcomprises a heavy chain variable region sequence of SEQ ID NO: 44 or 46,and a light chain variable region sequence of SEQ ID NO: 49, 50 or 51.In some embodiments, the antibody or antigen binding fragment thereofcomprises a heavy chain variable region sequence of SEQ ID NO: 46, and alight chain variable region sequence of SEQ ID NO: 51.

Another embodiment of the invention is an isolated antibody describedherein, or an antigen-binding fragment thereof, that binds to a PD-1protein, wherein the isolated antibody inhibits the binding ofbiotinylated EH12.2H7 antibody to Fc-PD-1 in a competition ELISA assay.Another embodiment is an isolated antibody described herein, or anantigen-binding fragment thereof, that binds to a PD-L1 protein, whereinthe isolated antibody inhibits the binding of biotinylated 29E2A3antibody to Fc-PD-L1 in a competition ELISA assay. Another embodiment isan isolated antibody described herein, or an antigen-binding fragmentthereof, that binds to a PD-L2 protein, wherein the isolated antibodyinhibits the binding of biotinylated 24F.10C12 antibody to Fc-PD-L2 in acompetition ELISA assay.

Another embodiment of the invention is an isolated antibody describedherein, or an antigen-binding fragment thereof, that binds to a PD-1protein, wherein the isolated antibody inhibits a PD-1-mediated signal.Another embodiment is an isolated antibody described herein, or anantigen-binding fragment thereof, that binds to a PD-L1 protein whereinthe isolated antibody inhibits a PD-L1-mediated signal. Anotherembodiment is an isolated antibody described herein, or anantigen-binding fragment thereof, that binds to a PD-L2 protein whereinthe isolated antibody inhibits a PD-L2-mediated signal.

In particular, an embodiment of the invention is an isolated nucleicacid encoding a polypeptide, wherein the polypeptide comprises asequence selected from the group consisting of SEQ ID NO: 25-51, or asequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical or homology to SEQ ID NO: 25-51.Another embodiment is a vector, host cell or animal comprising one ormore of these nucleic acids. Another aspect is a nucleic acid thathybridizes, under stringent conditions, with the complement of a nucleicacid encoding a polypeptide selected from the group consisting of SEQ IDNO: 25-51, or a sequence with at least about at least about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalhomology to SEQ ID NO: 25-51.

The invention also provides an isolated nucleic acid encoding a heavychain variable region and/or a light chain variable region of any of theantibodies or antigen binding-fragments thereof described herein. Insome embodiments, the nucleic acid is in a vector, such as an expressionvector. The invention also provides a host cell comprising one or morenucleic acids encoding the heavy and/or light chain of the antibodies orantigen-binding fragments described herein. In some embodiments, thehost cell produces the antibodies or antigen-binding fragments. Theinvention also provides methods of producing the antibody orantigen-binding fragment described herein, comprising culturing a cellthat produces the antibody or antigen-binding fragment, and recoveringthe antibody or antigen-binding fragment from the cell culture.

The invention further includes a pharmaceutical composition, comprisingan isolated antibody described herein, or an antigen-binding fragmentthereof, and a pharmaceutically-acceptable carrier.

The invention encompasses a method of reactivating an exhausted T cell,comprising contacting a population of T cells wherein at least somecells express PD-, PD-L1 and/or PD-L2 using an antibody described hereinor an antigen-binding fragment thereof either in vitro, ex vivo, or invivo.

The invention further pertains to a method of treating a subjectsuffering from a persistent infection, including a viral infection, abacterial infection, a helminth infection, or a protozoan infection,comprising administering to the subject a composition comprising aneffective amount of an isolated antibody described herein, or anantigen-binding fragment thereof.

The invention further encompasses a method of treating cancer,comprising administering to the subject a composition comprising aneffective amount of an isolated antibody described herein, or anantigen-binding fragment thereof, including wherein the isolatedantibody induces antibody-mediated cytotoxicity or is modified to induceantibody-mediated cytotoxicity or conjugated to an agent selected fromthe group consisting of a toxin and an imaging agent. In someembodiments, the antibody or the antigen-binding fragment that binds toa PD-L1 is administered to the subject having a cancer over-expressingPD-L1. In some embodiments, the antibody or the antigen-binding fragmentthat binds to a PD-L2 is administered to the subject having a cancerover-expressing PD-L2.

The invention further pertains to a method of treating a subjectsuffering from asthma, comprising administering to the subject acomposition comprising an effective amount of an isolated antibody thatbinds to a PD-L2 protein described herein, or an antigen-bindingfragment thereof.

The invention also encompasses a method of treating a subject sufferingfrom an inflammatory disease or transplant rejection, comprisingadministering to the subject a composition comprising an effectiveamount of an isolated antibody described herein, or an antigen-bindingfragment thereof, that binds to a PD-L1 protein or a PD-L2 protein.

The invention also encompasses an antibody, an antigen-binding fragmentor a polypeptide described herein for use in any of the methodsdescribed herein. The invention also encompasses the use of an antibody,an antigen-binding fragment or a polypeptide described herein for themanufacture of a medicament, such as a medicament for treating any ofthe diseases described herein in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of expression vectors used for cloningthe assembled human immunoglobulin sequences of the present invention.

FIG. 2A to FIG. 2E show composite, human heavy chain (FIG. 2A, VH1; FIG.2B, VH2; FIG. 2C, VH3; and FIG. 2D, VH4; FIG. 2E, VH5) variable regionsequences designed to correspond to that of the mouse anti-human PD-1antibody, EH12.2H7.

FIG. 3A to 3D show composite, human light chain (FIG. 3A, Vκ1; FIG. 3B,Vκ2; FIG. 3C, Vκ3; FIG. 3D, Vκ4) variable region sequences designed tocorrespond to that of the mouse anti-human PD-1 antibody, EH12.2H7.

FIG. 4A to FIG. 4E show composite, human heavy chain (FIG. 4A, VH1; FIG.4B, VH2; FIG. 4C, VH3; FIG. 4D, VH4; FIG. 4E, VH5) variable regionsequences designed to correspond to that of the mouse anti-human PD-L1antibody, 29E.2A3.

FIG. 5A to FIG. 5D show composite, human light chain (FIG. 5A, Vκ1; FIG.5B, Vκ2; FIG. 5C, Vκ3; FIG. 5D, Vκ4) variable region sequences designedto correspond to that of the mouse anti-human PD-L1 antibody, 29E.2A3.

FIG. 6A to FIG. 6E show composite, human heavy chain (FIG. 6A, VH1; FIG.6B, VH2; FIG. 6C, VH3; FIG. 6D, VH4; FIG. 6E, VH5) variable regionsequences designed to correspond to that of the mouse anti-human PD-L2antibody, 24F.10C12.

FIG. 7A to FIG. 7D show composite, human light chain (FIG. 7A, Vκ1; FIG.7B, Vκ2; FIG. 7C, Vκ3; FIG. 7D, Vκ4) variable region sequences designedto correspond to that of the mouse anti-human PD-L2 antibody, 24F.10C12.

FIG. 8A to FIG. 8C show SDS-PAGE results of 1 μg of composite, humanantibodies corresponding to the mouse anti-human antibodies, EH12.2H7(FIG. 8A), 29E.2A3 (FIG. 8B), and 24F.10C12 (FIG. 8C), respectively.

FIG. 9A to FIG. 9C show ELISA competition results of human antibodiescorresponding to and relative to the mouse anti-human antibodies,EH12.2H7, 29E.2A3, and 24F.10C12, respectively. In FIG. 9A, the bindingof the purified antibodies to human PD-1 was tested via competitionELISA. Varying concentrations of each antibody (0.06 μg/ml to 8 μg/ml)were mixed with a fixed concentration of biotinylated EH12.2H7 (40ng/ml) and bound to a PD-1 coated immulon maxisorb plate. Binding wasdetected via streptavidin-HRP and OPD substrate. Absorbance at 490 nmwas measured on a plate reader and this was plotted against the testantibody concentration. In FIG. 9B, the binding of the purifiedantibodies to human PD-L1 was tested via competition ELISA. Varyingconcentrations of each antibody (0.02 μg/ml to 8 μg/ml) were mixed witha fixed concentration of biotinylated 29E.2A3 (40 ng/ml) and bound to aPD-L1 coated immulon maxisorb plate. Binding was detected viastreptavidin-HRP and OPD substrate. Absorbance at 490 nm was measured ona plate reader and this was plotted against the test antibodyconcentration. In FIG. 9C, the binding of the purified antibodies tohuman PD-L2 was tested via competition ELISA. Varying concentrations ofeach antibody (0.02 μg/ml to 8 μg/ml) were mixed with a fixedconcentration of biotinylated 24F.10C12 (40 ng/ml) and bound to a PD-L2coated immulon maxisorb plate. Binding was detected via streptavidin-HRPand OPD substrate. Absorbance at 490 nm was measured on a plate readerand this was plotted against the test antibody concentration.

FIG. 10A to 10C show IC₅₀ binding data resulting from ELISA competitionanalysis of composite, human antibodies formed according to differentcombinations of composite, human heavy and light chains designed tocorrespond to those of the mouse anti-human antibodies, EH12.2H7 (FIG.10A), 29E.2A3 (FIG. 10B), and 24F.10C12 (FIG. 10C), respectively. Theassay was performed as described in FIG. 3. The IC₅₀ for eachcombination of heavy and light chain was normalized against the IC₅₀ ofthe mouse antibody. ND=No Data.

FIG. 11 shows the amino acid sequences of PD-1, PD-L1 and PD-L2.

FIG. 12 shows the amino acid sequences of the CDR regions of some of theComposite, Human Antibodies described herein.

FIG. 13 shows the amino acid sequences of the variable regions of someof the Composite, Human Antibodies described herein.

FIG. 14A and FIG. 14B shows effect of a humanized anti-PD-1 antibody anda humanized anti-PD-L1 antibody on the proliferative capacity of SIVGag-specific CD8 T cells in vitro. Each symbol represents an individualmacaque. Numbers in parenthesis represent fold increase in proliferationin the presence of a blocking Ab compared to no blocking Ab.

FIG. 15 shows that PD-L1 blockage restores antigen-driven proliferationof intrahepatic CD8 T cells (representative data from animal 1564).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel antibody-based therapeutics fortreating and diagnosing a variety of disorders mediated by PD-1, PD-L1,and/or PD-L2 (e.g., treatment of persistent infectious diseases, asthma,inflammatory diseases, transplant rejections and cancers).

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, the terms “PD-1”, “PD-L1”, and “PD-L2” include anyvariants or isoforms which are naturally expressed by cells, and/orfragments thereof having at least one biological activity of thefull-length polypeptide, unless otherwise expressly defined. Inaddition, the term “PD-1 ligand” includes either or both PD-L1 (Freemanet al. (2000) J. Exp. Med. 192:1027) and PD-L2 (Latchman et al. (2001)Nat. Immunol. 2:261) and any variants or isoforms which are naturallyexpressed by cells, and/or fragments thereof having at least onebiological activity of the full-length polypeptides. For example, PD-1,PD-L1, and PD-L2 sequences from different species, including humans, arewell known in the art (see, for example, herein incorporated in theirentirety by reference, Honjo et al., U.S. Pat. No. 5,629,204, whichdiscloses human and mouse PD-1 sequences; Wood et al., U.S. Pat. No.7,105,328, which discloses human PD-1 sequences; Chen et al., U.S. Pat.No. 6,803,192, which discloses human and mouse PD-L1 sequences; Wood etal., U.S. Pat. No. 7,105,328, which discloses human PD-L1 sequences;Freeman et al., US Pat. Pub. 20020164600, which discloses human andmouse PD-L2 sequences).

As used herein, the term “antibody” includes whole antibodies and anyantigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The V_(H) and V_(L) regions canbe further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen.“Inactivating antibodies” refers to antibodies that do not induce thecomplement system.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody-variable domain that are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology248:1-25 (Lo, ed., Human Press, Totowa, N. J., 2003)). Indeed, naturallyoccurring camelid antibodies consisting of a heavy chain only arefunctional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of hypervariable region delineations are in use and areencompassed herein. The Kabat Complementarity Determining Regions (CDRs)are based on sequence variability and are the most commonly used (Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)).Chothia refers instead to the location of the structural loops (Chothiaand Lesk J. Mol. Biol. 196:901-917 (1987)). The end of the ChothiaCDR-H1 loop when numbered using the Kabat numbering convention variesbetween H32 and H34 (see below) depending on the length of the loop(this is because the Kabat numbering scheme places the insertions atH35A and H35B; if neither 35A nor 35B is present, the loop ends at 32;if only 35A is present, the loop ends at 33; if both 35A and 35B arepresent, the loop ends at 34). The AbM hypervariable regions represent acompromise between the Kabat CDRs and Chothia structural loops, and areused by Oxford Molecular's AbM antibody modeling software. The “contact”hypervariable regions are based on an analysis of the available complexcrystal structures. The residues from each of these hypervariableregions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32, H30-H35B Kabat 33 or 34 Number- ing H1H31-H35 H26-H35 H26-H32 H30-H35 Chothia Number- ing H2 H50-H65 H50-H58H52-H56 H47-H58 H3 H95-H102 H95-H102 H95-H102 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in theVL and 26-35B (H1), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or95-102 (H3) in the VH. These extended hypervariable regions aretypically combinations of the Kabat and Chothia definitions, which mayoptionally further include residues identified using the Contactdefinition. The variable domain residues are numbered according to Kabatet al., supra for each of these definitions.

“Framework” or “FR” residues are those variable-domain residues otherthan the HVR residues as herein defined.

The expression “variable-domain residue-numbering as in Kabat” or“amino-acid-position numbering as in Kabat,” and variations thereof,refers to the numbering system used for heavy-chain variable domains orlight-chain variable domains of the compilation of antibodies in Kabatet al., supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy-chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy-chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue. Suitable native-sequence Fc regions foruse in the antibodies of the invention include human IgG1, IgG2 (IgG2A,IgG2B), IgG3 and IgG4.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors, FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see M. Daëron, Annu.Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet,Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995).Other FcRs, including those to be identified in the future, areencompassed by the term “FcR” herein.

The terms “CDR”, and its plural “CDRs”, refer to a complementaritydetermining region (CDR) of which three make up the binding character ofa light chain variable region (CDRL1, CDRL2 and CDRL3) and three make upthe binding character of a heavy chain variable region (CDRH1, CDRH2 andCDRH3). CDRs contribute to the functional activity of an antibodymolecule and are separated by amino acid sequences that comprisescaffolding or framework regions. The exact definitional CDR boundariesand lengths are subject to different classification and numberingsystems. CDRs may therefore be referred to by Kabat, Chothia, contact orany other boundary definitions, including the numbering system describedherein. Despite differing boundaries, each of these systems has somedegree of overlap in what constitutes the so called “hypervariableregions” within the variable sequences. CDR definitions according tothese systems may therefore differ in length and boundary areas withrespect to the adjacent framework region. See for example Kabat,Chothia, and/or MacCallum et al., (Kabat et al., in “Sequences ofProteins of Immunological Interest,” 5^(th) Edition, U.S. Department ofHealth and Human Services, 1992; Chothia et al., J. Mol. Biol., 1987,196: 901; and MacCallum et al., J. Mol. Biol., 1996, 262: 732, each ofwhich is incorporated by reference in its entirety).

As used herein, the term “antigen-binding portion” of an antibody (orsimply “antibody portion”), refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., PD-1, PD-L1, and/or PD-L2). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the V_(H), V_(L), CL andCH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; (iii) aFd fragment consisting of the V_(H) and CH1 domains; (iv) a Fv fragmentconsisting of the V_(H) and V_(L) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544 546),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR) or (vii) a combination of two or more isolatedCDRs which may optionally be joined by a synthetic linker. Furthermore,although the two domains of the Fv fragment, V_(H) and V_(L), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(H) and V_(L) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423 426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879 5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, orsyngeneic; or modified forms thereof (e.g., humanized, chimeric, etc.).Antibodies may also be fully human. Preferably, antibodies of theinvention bind specifically or substantially specifically to PD-1,PD-L1, or PD-L2 polypeptides. The term “monoclonal antibody” as usedherein, refers to an antibody which displays a single bindingspecificity and affinity for a particular epitope. Accordingly, the term“human monoclonal antibody” refers to an antibody which displays asingle binding specificity and which has variable and constant regionsderived from human germline or non-germline immunoglobulin sequences. Inone embodiment, human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic non-human animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene fused to an immortalized cell.

As used herein, the term an “isolated antibody” is intended to refer toan antibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to PD-1, PD-L1, or PD-L2 is substantially free ofantibodies that do not bind to PD-1, PD-L1, or PD-L2, respectively). Anisolated antibody that specifically binds to an epitope of PD-1, PD-L1,and/or PD-L2 may, however, have cross-reactivity to other PD-1, PD-L1,and/or PD-L2 proteins, respectively, from different species. However,the antibody preferably always binds to human PD-1, PD-L1, and/or PD-L2.In addition, an isolated antibody is typically substantially free ofother cellular material and/or chemicals. In one embodiment of theinvention, a combination of “isolated” monoclonal antibodies havingdifferent specificities to PD-1, PD-L1, and/or PD-L2 are combined in awell defined composition.

As used herein, the term “humanized antibody” refers to an antibody thatconsists of the CDR of antibodies derived from mammals other than human,and the FR region and the constant region of a human antibody. Ahumanized antibody is useful as an effective component in a therapeuticagent according to the present invention since antigenicity of thehumanized antibody in human body is lowered.

As used herein, the term “composite antibody” refers to an antibodywhich has variable regions comprising germline or non-germlineimmunoglobulin sequences from two or more unrelated variable regions.Additionally, the term “composite, human antibody” refers to an antibodywhich has constant regions derived from human germline or non-germlineimmunoglobulin sequences and variable regions comprising human germlineor non-germline sequences from two or more unrelated human variableregions. A composite, human antibody is useful as an effective componentin a therapeutic agent according to the present invention since theantigenicity of the composite, human antibody in human body is lowered.

As used herein, the term “recombinant human antibody” includes all humanantibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further in Section I,below), (b) antibodies isolated from a host cell transformed to expressthe antibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable and constant regionsderived from human germline and/or non-germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, the term “heterologous antibody” is defined in relationto the transgenic non-human organism producing such an antibody. Thisterm refers to an antibody having an amino acid sequence or an encodingnucleic acid sequence corresponding to that found in an organism notconsisting of the transgenic non-human animal, and generally from aspecies other than that of the transgenic non-human animal.

As used herein, the term “K_(D)” is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction.

As used herein, the term “specific binding” refers to antibody bindingto a predetermined antigen. Typically, the antibody binds with anaffinity (K_(D)) of approximately less than 10⁻⁷ M, such asapproximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower whendetermined by surface plasmon resonance (SPR) technology in a BIACORE3000 instrument using recombinant human PD-1, PD-L1, or PD-L2 as theanalyte and the antibody as the ligand, and binds to the predeterminedantigen with an affinity that is at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-,1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-,7.0-, 8.0-, 9.0-, or 10.0-fold or greater than its affinity for bindingto a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen”.

As used herein, the term “isotype” refers to the antibody class (e.g.,IgM or IgG1) that is encoded by heavy chain constant region genes.

As used herein, the term “glycosylation pattern” is defined as thepattern of carbohydrate units that are covalently attached to a protein,more specifically to an immunoglobulin protein. A glycosylation patternof a heterologous antibody can be characterized as being substantiallysimilar to glycosylation patterns which occur naturally on antibodiesproduced by the species of the nonhuman transgenic animal, when one ofordinary skill in the art would recognize the glycosylation pattern ofthe heterologous antibody as being more similar to said pattern ofglycosylation in the species of the nonhuman transgenic animal than tothe species from which the CH genes of the transgene were derived.

As used herein, the term “naturally-occurring” as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

As used herein, the term “rearranged” refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete V_(H) and V_(L) domain, respectively. Arearranged immunoglobulin gene locus can be identified by comparison togermline DNA; a rearranged locus will have at least one recombinedheptamer/nonamer homology element.

As used herein, the term “unrearranged” or “germline configuration” inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

As used herein, the term “isolated nucleic acid molecule” in referenceto nucleic acids encoding antibodies or antibody portions (e.g., V_(H),V_(L), CDR3) that bind to PD-1, PD-L1, or PD-L2, is intended to refer toa nucleic acid molecule in which the nucleotide sequences encoding theantibody or antibody portion are free of other nucleotide sequencesencoding antibodies or antibody portions that bind antigens other thanPD-1, PD-L1, or PD-L2, respectively, which other sequences may naturallyflank the nucleic acid in human genomic DNA. FIGS. 2-7 correspond to thenucleotide and amino acid sequences comprising the heavy chain (V_(H))and light chain (V_(L)) variable regions of the human anti-PD-1, PD-L1,or PD-L2 antibodies of the present invention, respectively.

The present invention also encompasses “conservative sequencemodifications” of the sequences set forth in the figures (e.g., FIGS.2-7), including nucleotide and amino acid sequence modifications whichdo not significantly affect or alter the binding characteristics of theantibody encoded by the nucleotide sequence or containing the amino acidsequence. Such conservative sequence modifications include nucleotideand amino acid substitutions, additions and deletions. Modifications canbe introduced into the sequence set forth in the figures (e.g., FIGS.2-7) by standard techniques known in the art, such as site-directedmutagenesis and PCR-mediated mutagenesis. Conservative amino acidsubstitutions include ones in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in a human anti-PD-1, anti-PD-L1, or anti-PD-L2antibody is preferably replaced with another amino acid residue from thesame side chain family.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a human anti-PD-1, PD-L1, or PD-L2antibody coding sequence, such as by saturation mutagenesis, and theresulting modified human anti-PD-1, anti-PD-L1, or anti-PD-L2 antibodiescan be screened for binding activity.

Accordingly, antibodies encoded by the heavy and light chain variableregion nucleotide sequences disclosed herein and/or containing the heavyand light chain variable region amino acid sequences disclosed herein(e.g., FIGS. 2-7) include substantially similar antibodies encoded by orcontaining similar sequences which have been conservatively modified.Further discussion as to how such substantially similar antibodies canbe generated based on the sequences (i.e., heavy and light chainvariable regions) disclosed herein (e.g., FIGS. 2-7) is provided below.

In addition, there is a known and definite correspondence between theamino acid sequence of a particular protein and the nucleotide sequencesthat can code for the protein, as defined by the genetic code (shownbelow). Likewise, there is a known and definite correspondence betweenthe nucleotide sequence of a particular nucleic acid and the amino acidsequence encoded by that nucleic acid, as defined by the genetic code.

GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R)AGA, ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AATAspartic acid (Asp, D) GAC, GAT Cysteine (Cys, C) TGC, TGTGlutamic acid (Glu, E) GAA, GAG Glutamine (Gln, Q) CAA, CAGGlycine (Gly, G) GGA, GGC, GGG, GGT Histidine (His, H) CAC, CATIsoleucine (Ile, I) ATA, ATC, ATT Leucine (Leu, L)CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAGMethionine (Met, M) ATG Phenylalanine (Phe, F) TTC, TTT Proline (Pro, P)CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCTThreonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGGTyrosine (Tyr, Y) TAC, TAT Valine (Val, V) GTA, GTC, GTG, GTTTermination signal TAA, TAG, TGA (end)

An important and well known feature of the genetic code is itsredundancy, whereby, for most of the amino acids used to make proteins,more than one coding nucleotide triplet may be employed (illustratedabove). Therefore, a number of different nucleotide sequences may codefor a given amino acid sequence. Such nucleotide sequences areconsidered functionally equivalent since they result in the productionof the same amino acid sequence in all organisms (although certainorganisms may translate some sequences more efficiently than they doothers). Moreover, occasionally, a methylated variant of a purine orpyrimidine may be found in a given nucleotide sequence. Suchmethylations do not affect the coding relationship between thetrinucleotide codon and the corresponding amino acid.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, or more of the nucleotides, andmore preferably at least about 97%, 98%, 99% or more of the nucleotides.Alternatively, substantial homology exists when the segments willhybridize under selective hybridization conditions, to the complement ofthe strand.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available on theworld wide web at the GCG company website), using a NWSgapdna.CMP matrixand a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,3, 4, 5, or 6. The percent identity between two nucleotide or amino acidsequences can also be determined using the algorithm of E. Meyers and W.Miller (CABIOS, 4:11 17 (1989)) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. In addition, the percentidentity between two amino acid sequences can be determined using theNeedleman and Wunsch (J. Mol. Biol. (48):444 453 (1970)) algorithm whichhas been incorporated into the GAP program in the GCG software package(available on the world wide web at the GCG company website), usingeither a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403 10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389 3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used (available on the world wide web at the NCBI website).

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures thereof may be mutated, inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

As used herein, the term “vector” is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

As used herein, the term “recombinant host cell” (or simply “hostcell”), is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions of the presentinvention can be used to treat a subject with an inflammatory disease,such as arthritis, e.g., rheumatoid arthritis. The term “non-humananimal” includes all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, dog, cow, chickens, amphibians, reptiles,etc.

As used herein, the term “modulate” includes up-regulation anddown-regulation, e.g., enhancing or inhibiting a response.

As used herein, the term “inhibit” includes the decrease, limitation, orblockage, of, for example a particular action, function, or interaction.

As used herein, the term “immune cell” refers to cells that play a rolein the immune response Immune cells are of hematopoietic origin, andinclude lymphocytes, such as B cells and T cells; natural killer cells;myeloid cells, such as monocytes, macrophages, eosinophils, mast cells,basophils, and granulocytes.

As used herein, the term “T cell” includes CD4+ T cells and CD8+ Tcells. The term T cell also includes T helper 1 type T cells, T helper 2type T cells, T helper 17 type T cells and inhibitory T cells. The term“antigen presenting cell” includes professional antigen presenting cells(e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) aswell as other antigen presenting cells (e.g., keratinocytes, endothelialcells, astrocytes, fibroblasts, oligodendrocytes).

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune responses that are influenced bymodulation of T cell costimulation. Exemplary immune responses include Tcell responses, e.g., cytokine production, and cellular cytotoxicity. Inaddition, the term immune response includes immune responses that areindirectly affected by T cell activation, e.g., antibody production(humoral responses) and activation of cytokine responsive cells, e.g.,macrophages.

As used herein, the term “costimulate,” as used with reference toactivated immune cells, includes the ability of a costimulatorypolypeptide to provide a second, non-activating receptor mediated signal(a “costimulatory signal”) that induces proliferation and/or effectorfunction. For example, a costimulatory signal can result in cytokinesecretion, e.g., in a T cell that has received a Tcell-receptor-mediated signal Immune cells that have received acell-receptor mediated signal, e.g., via an activating receptor arereferred to herein as “activated immune cells.”

As used herein, the term “inhibitory signal” refers to a signaltransmitted via an inhibitory receptor (e.g., CTLA4 or PD-1) for apolypeptide on an immune cell. Such a signal antagonizes a signal via anactivating receptor (e.g., via a TCR or CD3 polypeptide) and can resultin, e.g., inhibition of second messenger generation; an inhibition ofproliferation; an inhibition of effector function in the immune cell,e.g., reduced phagocytosis, reduced antibody production, reducedcellular cytotoxicity, the failure of the immune cell to producemediators, (such as cytokines (e.g., IL-2) and/or mediators of allergicresponses); or the development of anergy.

As used herein, the term “unresponsiveness” includes refractivity ofimmune cells to stimulation, e.g., stimulation via an activatingreceptor or a cytokine. Unresponsiveness can occur, e.g., because ofexposure to immunosuppressants or exposure to high doses of antigen. Asused herein, the term “anergy” or “tolerance” includes refractivity toactivating receptor-mediated stimulation. Such refractivity is generallyantigen-specific and persists after exposure to the tolerizing antigenhas ceased. For example, anergy in T cells (as opposed tounresponsiveness) is characterized by lack of cytokine production, e.g.,IL-2. T cell anergy occurs when T cells are exposed to antigen andreceive a first signal (a T cell receptor or CD-3 mediated signal) inthe absence of a second signal (a costimulatory signal). Under theseconditions, reexposure of the cells to the same antigen (even ifreexposure occurs in the presence of a costimulatory polypeptide)results in failure to produce cytokines and, thus, failure toproliferate. Anergic T cells can, however, proliferate if cultured withcytokines (e.g., IL-2). For example, T cell anergy can also be observedby the lack of IL-2 production by T lymphocytes as measured by ELISA orby a proliferation assay using an indicator cell line. Alternatively, areporter gene construct can be used. For example, anergic T cells failto initiate IL-2 gene transcription induced by a heterologous promoterunder the control of the 5′ IL-2 gene enhancer or by a multimer of theAP1 sequence that can be found within the enhancer (Kang et al. (1992)Science 257:1134).

As used herein, the term “activity,” when used with respect to apolypeptide, e.g., PD-1, PD-L1, or PD-L2 polypeptide, includesactivities which are inherent in the structure of the protein. Forexample, with regard to PD-1 ligand, the term “activity” includes theability to modulate immune cell costimulation (e.g. by modulating acostimulatory signal in an activated immune cell) or to modulateinhibition by modulating an inhibitory signal in an immune cell (e.g.,by engaging a natural receptor on an immune cell). Those of skill in theart will recognize that when a PD-1 ligand polypeptide binds to acostimulatory receptor, a costimulatory signal can be generated in theimmune cell. When a PD-1 ligand polypeptide binds to an inhibitoryreceptor, an inhibitory signal is generated in the immune cell. Also,when a PD-1 ligand binds to a B7-1 polypeptide, an inhibitory signal canbe generated (Butte et al. (2007) Immunity 27:111).

With respect to PD-1, the term “activity” includes the ability of a PD-1polypeptide to modulate an inhibitory signal in an immune cell, e.g., byengaging a natural PD-1 ligand on an antigen presenting cell. PD-1transmits an inhibitory signal to an immune cell in a manner similar toCTLA4. Modulation of an inhibitory signal in an immune cell results inmodulation of proliferation of, and/or cytokine secretion by, an immunecell. Thus, the term “PD-1 activity” includes the ability of a PD-1polypeptide to bind its natural ligand(s), the ability to modulateimmune cell costimulatory or inhibitory signals, and the ability tomodulate the immune response.

As used herein, the term “interaction”, when referring to an interactionbetween two molecules, refers to the physical contact (e.g., binding) ofthe molecules with one another. Generally, such an interaction resultsin an activity (which produces a biological effect) of one or both ofsaid molecules. The activity may be a direct activity of one or both ofthe molecules, (e.g., signal transduction). Alternatively, one or bothmolecules in the interaction may be prevented from binding a ligand, andthus be held inactive with respect to ligand binding activity (e.g.,binding its ligand and triggering or inhibiting costimulation). Toinhibit such an interaction results in the disruption of the activity ofone or more molecules involved in the interaction. To enhance such aninteraction is to prolong or increase the likelihood of said physicalcontact, and prolong or increase the likelihood of said activity.

As used herein the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise.

It is understood that aspects and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode polypeptides of the present invention (e.g., those in FIGS.2-7) or biologically active portions thereof, as well as nucleic acidfragments sufficient for use as hybridization probes to identify nucleicacid molecules encoding these polypeptides and fragments for use as PCRprimers for the amplification or mutation of the nucleic acid molecules.As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA)and analogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acidmolecule is free of sequences which naturally flank the nucleic acid(i.e., sequences located at the 5′ and 3′ ends of the nucleic acidmolecule) in the genomic DNA of the organism from which the nucleic acidis derived. For example, an “isolated” nucleic acid molecule, such as acDNA molecule, can be substantially free of other cellular material, orculture medium, when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized.

A nucleic acid molecule of the present invention (e.g., those in FIGS.2-7), or a portion thereof, can be isolated using standard molecularbiology techniques and the sequence information provided herein. Forexample, a nucleic acid molecule encompassing all or a portion ofsequences shown in FIGS. 2-7 can be isolated by the polymerase chainreaction (PCR) using synthetic oligonucleotide primers designed basedupon the sequences shown in FIGS. 2-7.

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA or, alternatively, genomic DNA as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid molecule so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to nucleic acid sequences ofthe invention can be prepared by standard synthetic techniques, e.g.,using an automated DNA synthesizer.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of anucleic acid molecule of the present invention (e.g., those in FIGS.2-7), or a portion thereof. A nucleic acid molecule which iscomplementary to a nucleic acid molecule of the present invention (e.g.,those in FIGS. 2-7), or a portion thereof, is one which is sufficientlycomplementary to the nucleotide sequence shown in FIGS. 2-7, such thatit can hybridize to the respective nucleotide sequence shown in FIGS.2-7, thereby forming a stable duplex.

In still another embodiment, an isolated nucleic acid molecule of thepresent invention comprises a nucleotide sequence which is at leastabout 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the entire length of the nucleotide sequenceshown in FIGS. 2-7, or a portion of any of these nucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of a nucleic acid molecule of the present invention (e.g., thosein FIGS. 2-7), or a portion thereof, for example, a fragment which canbe used as a probe or primer or a fragment which encodes a portion of apolypeptide of the invention, e.g., those in FIGS. 2-7. The probe/primertypically comprises substantially purified oligonucleotide. Theoligonucleotide typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12 or 15,preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55,60, 65, or 75 consecutive nucleotides of a nucleic acid molecule of thepresent invention (e.g., those in FIGS. 2-7); of an anti-sense sequenceof a nucleic acid molecule of the present invention (e.g., those inFIGS. 2-7); or of a mutant of a nucleic acid molecule of the presentinvention (e.g., those in FIGS. 2-7).

Probes based on a nucleic acid molecule of the present invention (e.g.,those in FIGS. 2-7) can be used to detect transcripts or genomicsequences encoding the same or homologous polypeptides. In oneembodiment, the probe further comprises a label group attached thereto,e.g., the label group can be a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor.

A nucleic acid fragment encoding a “biologically active portion of apolypeptide of the invention” can be prepared by isolating a portion ofthe nucleotide sequence of a nucleic acid molecule of the presentinvention (e.g., those in FIGS. 2-7) which encodes a polypeptide havinga biological activity of a polypeptide of the invention (e.g., theability to bind to its antigenic target), expressing the encoded portionof the polypeptide of the invention (e.g., by recombinant expression invitro) and assessing the activity of the encoded portion of thepolypeptide of the invention.

The invention further encompasses nucleic acid molecules that differfrom nucleotide sequence(s) shown in FIGS. 2-7 due to degeneracy of thegenetic code and thus encode the same polypeptides as those encoded bythe respective nucleotide sequence shown in FIGS. 2-7. In anotherembodiment, an isolated nucleic acid molecule of the invention has anucleotide sequence encoding a polypeptide of the present invention(e.g., those in FIGS. 2-7).

Nucleic acid molecules corresponding to homologues of a nucleic acidmolecule of the present invention (e.g., those in FIGS. 2-7) can beisolated based on their homology to the nucleic acids disclosed hereinusing the cDNAs disclosed herein, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15, 20, 25, 30 or more nucleotides in lengthand hybridizes under stringent conditions to the nucleic acid moleculecomprising a nucleic acid molecule of the present invention (e.g., thosein FIGS. 2-7).

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences that are significantly identical orhomologous to each other remain hybridized to each other. Preferably,the conditions are such that sequences at least about 70%, morepreferably at least about 80%, even more preferably at least about 85%or 90% identical to each other remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additionalstringent conditions can be found in Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), chapters 7, 9 and 11. A non-limiting example of stringenthybridization conditions includes hybridization in 4× or 6× sodiumchloride/sodium citrate (SSC), at about 65-70° C. (or hybridization in4×SSC plus 50% formamide at about 42-50° C.) followed by one or morewashes in 1×SSC, at about 65-70° C. A further non-limiting example ofstringent hybridization conditions includes hybridization at 6×SSC at45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C. Anon-limiting example of highly stringent hybridization conditionsincludes hybridization in 1×SSC, at about 65-70° C. (or hybridization in1×SSC plus 50% formamide at about 42-50° C.) followed by one or morewashes in 0.3×SSC, at about 65-70° C. A non-limiting example of reducedstringency hybridization conditions includes hybridization in 4× or6×SSC, at about 50-60° C. (or alternatively hybridization in 6×SSC plus50% formamide at about 40-45° C.) followed by one or more washes in 2×,at about 50-60° C. Ranges intermediate to the above-recited values,e.g., at 65-70° C. or at 42-50° C. are also intended to be encompassedby the present invention. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH₂PO₄, and1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCland 15 mM sodium citrate) in the hybridization and wash buffers; washesare performed for 15 minutes each after hybridization is complete. Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature (T_(m)) of the hybrid, where T_(m) is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, T_(m) (° C.)=2(# of A+T bases)+4(# of G+C bases). For hybridsbetween 18 and 49 base pairs in length, T_(m) (° C.)=81.5+16.6(log₁₀[Na⁺])+0.41 (% G+C)−(600/N), where N is the number of bases in thehybrid, and [Na⁺] is the concentration of sodium ions in thehybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional non-limitingexample of stringent hybridization conditions is hybridization in0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed by one or morewashes at 0.02M NaH₂PO₄, 1% SDS at 65° C., see e.g., Church and Gilbert(1984) Proc. Natl. Acad. Sci. USA 81:1991-1995 (or alternatively0.2×SSC, 1% SDS).

The skilled artisan will further appreciate that changes can beintroduced by mutation into a nucleic acid molecule of the presentinvention (e.g., those in FIGS. 2-7), thereby leading to changes in theamino acid sequence of the encoded polypeptides of the presentinvention, without altering the functional ability of the polypeptides.For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues can be made in anucleic acid molecule of the present invention (e.g., those in FIGS.2-7). A “non-essential” amino acid residue is a residue that can bealtered from a nucleic acid molecule of the present invention (e.g.,those in FIGS. 2-7) without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity. Forexample, amino acid residues that are conserved among the polypeptidesof the present invention, e.g., those required for binding of thepolypeptides to its target antigen, are predicted to be particularlyunamenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding polypeptides of the present invention (e.g., those inFIGS. 2-7) that contain changes in amino acid residues that are notessential for activity. Such polypeptides differ in amino acid sequencefrom those in FIGS. 2-7, yet retain biological activity. In oneembodiment, the isolated nucleic acid molecule comprises a nucleotidesequence encoding a polypeptide, wherein the polypeptide comprises anamino acid sequence at least about 71%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to those in FIGS.2-7.

An isolated nucleic acid molecule encoding a polypeptide identical tothe polypeptides of those in FIGS. 2-7 can be created by introducing oneor more nucleotide substitutions, additions or deletions into thenucleotide sequence of those in FIGS. 2-7 such that one or more aminoacid substitutions, additions or deletions are introduced into theencoded polypeptide. Mutations can be introduced into nucleic acidmolecules of the present invention (e.g., those in FIGS. 2-7) bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. In one embodiment, conservative amino acid substitutionsare made at one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a polypeptide of theinvention (e.g., those in FIGS. 2-7) can be replaced with another aminoacid residue from the same side chain family. Alternatively, in anotherembodiment, mutations can be introduced randomly along all or part of anucleic acid molecule(s) of the present invention (e.g., those in FIGS.2-7), such as by saturation mutagenesis, and the resultant mutants canbe screened for biological activity to identify mutants that retainactivity. Following mutagenesis of a nucleic acid molecule of thepresent invention (e.g., those in FIGS. 2-7), the encoded polypeptidecan be expressed recombinantly and the activity of the polypeptide canbe determined.

In one embodiment, a mutant polypeptide of the invention can be assayedfor the ability to bind to and/or modulate the activity of a naturalPD-1 (e.g., PD-1 ligands) or PD-1 ligand partner (e.g., PD-1 and B7-1),modulate intra- or intercellular signaling, modulate activation of Tlymphocytes, and/or modulate the immune response of an organism.

Yet another aspect of the invention pertains to isolated nucleic acidmolecules encoding fusion proteins. Such nucleic acid molecules,comprising at least a first nucleotide sequence encoding a polypeptideof the invention (e.g., those in FIGS. 2-7) operatively linked to asecond nucleotide sequence encoding a polypeptide of the invention(e.g., those in FIGS. 2-7) can be prepared by standard recombinant DNAtechniques.

The expression characteristics of a nucleic acid molecules of thepresent invention (e.g., those in FIGS. 2-7) within a cell line ormicroorganism may be modified by inserting a heterologous DNA regulatoryelement into the genome of a stable cell line or cloned microorganismsuch that the inserted regulatory element is operatively linked with thea nucleic acid molecules of the present invention (e.g., those in FIGS.2-7). For example, a heterologous regulatory element may be insertedinto a stable cell line or cloned microorganism, such that it isoperatively linked with a nucleic acid molecules of the presentinvention (e.g., those in FIGS. 2-7), using techniques, such as targetedhomologous recombination, which are well known to those of skill in theart, and described, e.g., in Chappel, U.S. Pat. No. 5,272,071; PCTpublication No. WO 91/06667, published May 16, 1991.

II. Isolated Polypeptide Molecules

One aspect of the invention pertains to isolated polypeptides of thepresent invention (including antibodies and antigen-binding fragmentsthereof described herein, and those in FIGS. 2-7), and biologicallyactive portions thereof. In one embodiment, polypeptides of the presentinvention (e.g., those in FIGS. 2-7), and biologically active portionsthereof can be isolated from cells or tissue sources by an appropriatepurification scheme using standard protein purification techniques. Inanother embodiment, polypeptides of the present invention (e.g., thosein FIGS. 2-7), and biologically active portions thereof are produced byrecombinant DNA techniques. Alternatively, polypeptides of the presentinvention (e.g., those in FIGS. 2-7), and biologically active portionsthereof can be chemically synthesized using standard peptide synthesistechniques.

An “isolated” or “purified” polypeptide or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which thepolypeptides of the present invention (e.g., those in FIGS. 2-7) isderived, or substantially free from chemical precursors or otherchemicals when chemically synthesized. The language “substantially freeof cellular material” includes preparations of polypeptide(s) of thepresent invention (e.g., those in FIGS. 2-7), and biologically activeportions thereof, in which the polypeptide is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of polypeptide(s) of thepresent invention (e.g., those in FIGS. 2-7), and biologically activeportions thereof having less than about 30% (by dry weight) of proteinsnot of the present invention (also referred to herein as a“contaminating protein”), more preferably less than about 20% ofproteins not of the present invention, still more preferably less thanabout 10% of proteins not of the present invention, and most preferablyless than about 5% of proteins not of the present invention. Whenpolypeptides of the present invention (e.g., those in FIGS. 2-7) orbiologically active portion thereof are recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of polypeptide(s) of the presentinvention (e.g., those in FIGS. 2-7) or biologically active portionthereof in which the polypeptide is separated from chemical precursorsor other chemicals which are involved in the synthesis of thepolypeptide. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations ofpolypeptide(s) of the present invention (e.g., those in FIGS. 2-7) orbiologically active portion thereof having less than about 30% (by dryweight) of chemical precursors or of proteins not of the presentinvention, more preferably less than about 20% chemical precursors or ofproteins not of the present invention, still more preferably less thanabout 10% chemical precursors or of proteins not of the presentinvention, and most preferably less than about 5% chemical precursors orof proteins not of the present invention.

As used herein, a “biologically active portion” of polypeptide(s) of thepresent invention (e.g., those in FIGS. 2-7) include polypeptides whichparticipates in an interaction between PD-1 and a non-PD-1 molecule,PD-L1 and a non-PD-L1 molecule, or PD-L2 and a non-PD-L2 molecule, e.g.,a natural ligand of PD-1, e.g., PD-1 ligands, or a natural ligand ofPD-1 ligands, e.g., PD-1 or B7-1, respectively. Biologically activeportions of a polypeptide(s) of the present invention (e.g., those inFIGS. 2-7) include peptides comprising amino acid sequences sufficientlyidentical to or derived from the amino acid sequence of polypeptide(s)of the present invention (e.g., those in FIGS. 2-7), which include feweramino acids than the respective, full length polypeptide(s) of thepresent invention (e.g., those in FIGS. 2-7), and exhibit at least oneactivity of the respective polypeptide(s) of the present invention(e.g., those in FIGS. 2-7). In one embodiment, biologically activeportions comprise a domain or motif with the ability to specificallybind PD-1 or a PD-L1 ligand according to the antigen, respectively, towhich it was raised or designed to bind. Biologically active portions ofpolypeptide(s) of the present invention (e.g., those in FIGS. 2-7) canbe used as targets for developing agents which modulate an activitymediated by PD-1, PD-L1, or PD-L2, e.g., immune cell activation orsuppression.

In another embodiment, polypeptide(s) of the present invention (e.g.,those in FIGS. 2-7) has an amino acid sequence shown in FIGS. 2-7. Inother embodiments, the polypeptide is substantially identical topolypeptide(s) shown in FIGS. 2-7, and retains the functional activityof the respective polypeptide(s) shown in FIGS. 2-7, yet differs inamino acid sequence due to mutagenesis, as described in detail insubsection I above. Accordingly, in another embodiment, a polypeptide(s)of the present invention is a polypeptide which comprises an amino acidsequence at least about 71%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96% 97%, 98%, 99%, 99.5%, or 99.9% or more identical to apolypeptide(s) shown in FIGS. 2-7.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Inone embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%,or 99.9% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The invention also provides chimeric or fusion proteins. As used herein,a “chimeric protein” or “fusion protein” comprises a polypeptide(s) ofthe present invention (e.g., those in FIGS. 2-7) operatively linked to apolypeptide not of the present invention. A “polypeptide(s) of thepresent invention” refers to a polypeptide having an amino acid sequencecorresponding to a polypeptide shown in FIGS. 2-7, whereas a“polypeptide not of the present invention” refers to a polypeptide nothaving an amino acid sequence corresponding to a polypeptide which isnot substantially homologous to a polypeptide shown in FIGS. 2-7, e.g.,a polypeptide which is different from a polypeptide shown in FIGS. 2-7and which is derived from the same or a different organism. Within thefusion protein, the term “operatively linked” is intended to indicatethat the polypeptide(s) of the present invention and the polypeptide(s)not of the present invention are fused in-frame to each other. Thepolypeptide(s) not of the present invention can be fused to theN-terminus or C-terminus of the polypeptide(s) of the present inventionand corresponds to a moiety that alters the solubility, bindingaffinity, stability, or valency of the polypeptide(s) of the presentinvention.

For example, in one embodiment, the fusion protein is a GST fusionprotein with a polypeptide(s) of the present invention. Such fusionproteins can facilitate the purification of recombinant polypeptides ofthe invention. In another embodiment, the fusion protein contains aheterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion ofpolypeptide(s) of the present invention can be increased through use ofa heterologous signal sequence.

A chimeric or fusion polypeptide(s) of the present invention (e.g.,those in FIGS. 2-7) can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the differentpolypeptide sequences are ligated together in-frame in accordance withconventional techniques, for example by employing blunt-ended orstagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, Ausubel et al.,eds., John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide).

The amino acid sequences of polypeptide(s) of the present invention(e.g., those in FIGS. 2-7) identified herein will enable those of skillin the art to produce polypeptides corresponding to polypeptide(s) ofthe present invention (e.g., those in FIGS. 2-7). Such polypeptides canbe produced in prokaryotic or eukaryotic host cells by expression ofpolynucleotides encoding a polypeptide(s) of the present invention(e.g., those in FIGS. 2-7. Alternatively, such peptides can besynthesized by chemical methods. Methods for expression of heterologouspolypeptides in recombinant hosts, chemical synthesis of polypeptides,and in vitro translation are well known in the art and are describedfurther in Maniatis et al., Molecular Cloning: A Laboratory Manual(1989), 2nd Ed., Cold Spring Harbor, N. Y.; Berger and Kimmel, Methodsin Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987),Academic Press, Inc., San Diego, Calif.; Merrifield, J. (1969) J. Am.Chem. Soc. 91:501; Chaiken I. M. (1981) CRC Crit. Rev. Biochem. 11:255;Kaiser et al. (1989) Science 243:187; Merrifield, B. (1986) Science232:342; Kent, S. B. H. (1988) Annu. Rev. Biochem. 57:957; and Offord,R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which areincorporated herein by reference).

III. Antibodies to PD-1, PD-L1, and/or PD-L2

Antibodies to PD-1, PD-L1, or PD-L2 described herein may be producedusing any methods described herein or known in the art. Monoclonalantibodies (e.g., human antibodies) of the invention can be producedusing a variety of known techniques, such as the standard somatic cellhybridization technique described by Kohler and Milstein, Nature 256:495 (1975). Although somatic cell hybridization procedures arepreferred, in principle, other techniques for producing monoclonalantibodies also can be employed, e.g., viral or oncogenic transformationof B lymphocytes, phage display technique using libraries of humanantibody genes.

One method for generating hybridomas which produce monoclonal antibodiesof the invention is the murine system. Hybridoma production in the mouseis well known in the art, including immunization protocols andtechniques for isolating and fusing immunized splenocytes.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a polypeptide immunogen. The polypeptide antibodytiter in the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized polypeptide. If desired, the antibody directed againstthe antigen can be isolated from the mammal (e.g., from the blood) andfurther purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. 76:2927-31; and Yeh et al. (1982) Int. J.Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique(Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generallyKenneth, R. H. in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); Lerner, E. A.(1981) Yale J. Biol. Med. 54:387-402; Gefter, M. L. et al. (1977)Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with an immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds to the polypeptideantigen, preferably specifically.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-PD-1, PD-L1, or PD-L2 monoclonal antibody (see, e.g., Galfre, G. etal. (1977) Nature 266:55052; Gefter et al. (1977) supra; Lerner (1981)supra; Kenneth (1980) supra). Moreover, the ordinary skilled worker willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line. Preferred immortal cell lines aremouse myeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O—Ag14 myeloma lines. These myeloma lines are available from theAmerican Type Culture Collection (ATCC), Rockville, Md. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bind agiven polypeptide, e.g., using a standard ELISA assay.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal specific for one of the above described polypeptides can beidentified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withthe appropriate polypeptide to thereby isolate immunoglobulin librarymembers that bind the polypeptide. Kits for generating and screeningphage display libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening an antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.International Publication No. WO 92/18619; Dower et al. InternationalPublication No. WO 91/17271; Winter et al. International Publication WO92/20791; Markland et al. International Publication No. WO 92/15679;Breitling et al. International Publication WO 93/01288; McCafferty etal. International Publication No. WO 92/01047; Garrard et al.International Publication No. WO 92/09690; Ladner et al. InternationalPublication No. WO 90/02809; Fuchs et al. (1991) Biotechnology (NY)9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J.12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clarkson etal. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci.USA 89:3576-3580; Garrard et al. (1991) Biotechnology (NY) 9:1373-1377;Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et al.(1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al.(1990) Nature 348:552-554.

Additionally, recombinant anti-PD-1, PD-L1, or PD-L2 antibodies, such aschimeric, composite, and humanized monoclonal antibodies, which can bemade using standard recombinant DNA techniques, can be generated. Suchchimeric, composite, and humanized monoclonal antibodies can be producedby recombinant DNA techniques known in the art, for example usingmethods described in Robinson et al. International Patent PublicationPCT/US86/02269; Akira et al. European Patent Application 184,187;Taniguchi, M. European Patent Application 171,496; Morrison et al.European Patent Application 173,494; Neuberger et al. PCT Application WO86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.European Patent Application 125,023; Better et al. (1988) Science240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987) CancerRes. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al.(1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985)Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S.Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan etal. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

In addition, humanized antibodies can be made according to standardprotocols such as those disclosed in U.S. Pat. No. 5,565,332. In anotherembodiment, antibody chains or specific binding pair members can beproduced by recombination between vectors comprising nucleic acidmolecules encoding a fusion of a polypeptide chain of a specific bindingpair member and a component of a replicable generic display package andvectors containing nucleic acid molecules encoding a second polypeptidechain of a single binding pair member using techniques known in the art,e.g., as described in U.S. Pat. Nos. 5,565,332, 5,871,907, or 5,733,743.The use of intracellular antibodies to inhibit protein function in acell is also known in the art (see e.g., Carlson, J. R. (1988) Mol.Cell. Biol. 8:2638-2646; Biocca, S. et al. (1990) EMBO J. 9:101-108;Werge, T. M. et al. (1990) FEBS Lett. 274:193-198; Carlson, J. R. (1993)Proc. Natl. Acad. Sci. USA 90:7427-7428; Marasco, W. A. et al. (1993)Proc. Natl. Acad. Sci. USA 90:7889-7893; Biocca, S. et al. (1994)Biotechnology (NY) 12:396-399; Chen, S-Y. et al. (1994) Hum. Gene Ther.5:595-601; Duan, L et al. (1994) Proc. Natl. Acad. Sci. USA91:5075-5079; Chen, S-Y. et al. (1994) Proc. Natl. Acad. Sci. USA91:5932-5936; Beerli, R. R. et al. (1994) J. Biol. Chem.269:23931-23936; Beerli, R. R. et al. (1994) Biochem. Biophys. Res.Commun. 204:666-672; Mhashilkar, A. M. et al. (1995) EMBO J.14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad. Sci. USA92:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al.).

In another embodiment, human monoclonal antibodies directed againstPD-1, PD-L1, or PD-L2 can be generated using transgenic ortranschromosomal mice carrying parts of the human immune system ratherthan the mouse system. In one embodiment, transgenic mice, referred toherein as “HuMAb mice” which contain a human immunoglobulin geneminiloci that encodes unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg, N. et al. (1994)Nature 368(6474): 856 859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed inLonberg, N. (1994) Handbook of Experimental Pharmacology 113:49 101;Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65 93,and Harding, F. and Lonberg, N. (1995) Ann. N. Y Acad. Sci 764:536 546).The preparation of HuMAb mice is described in Taylor, L. et al. (1992)Nucleic Acids Research 20:6287 6295; Chen, J. et al. (1993)International Immunology 5: 647 656; Tuaillon et al. (1993) Proc. Natl.Acad. Sci USA 90:3720 3724; Choi et al. (1993) Nature Genetics 4:117123; Chen, J. et al. (1993) EMBO J. 12: 821 830; Tuaillon et al. (1994)J. Immunol. 152:2912 2920; Lonberg et al., (1994) Nature 368(6474): 856859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49101; Taylor, L. et al. (1994) International Immunology 6: 579 591;Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65 93;Harding, F. and Lonberg, N. (1995) Ann N.Y. Acad. Sci 764:536 546;Fishwild, D. et al. (1996) Nature Biotechnology 14: 845 851. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429;all to Lonberg and Kay, and GenPharm International; U.S. Pat. No.5,545,807 to Surani et al.; International Publication Nos. WO 98/24884,published on Jun. 11, 1998; WO 94/25585, published Nov. 10, 1994; WO93/1227, published Jun. 24, 1993; WO 92/22645, published Dec. 23, 1992;WO 92/03918, published Mar. 19, 1992.

In another embodiment, an antibody for use in the invention is abispecific antibody. A bispecific antibody has binding sites for twodifferent antigens within a single antibody polypeptide. Antigen bindingmay be simultaneous or sequential. Triomas and hybrid hybridomas are twoexamples of cell lines that can secrete bispecific antibodies. Examplesof bispecific antibodies produced by a hybrid hybridoma or a trioma aredisclosed in U.S. Pat. No. 4,474,893. Bispecific antibodies have beenconstructed by chemical means (Staerz et al. (1985) Nature 314:628, andPerez et al. (1985) Nature 316:354) and hybridoma technology (Staerz andBevan (1986) Proc. Natl. Acad. Sci. USA, 83:1453, and Staerz and Bevan(1986) Immunol. Today 7:241). Bispecific antibodies are also describedin U.S. Pat. No. 5,959,084. Fragments of bispecific antibodies aredescribed in U.S. Pat. No. 5,798,229. Bispecific agents can also begenerated by making heterohybridomas by fusing hybridomas or other cellsmaking different antibodies, followed by identification of clonesproducing and co-assembling both antibodies. They can also be generatedby chemical or genetic conjugation of complete immunoglobulin chains orportions thereof such as Fab and Fv sequences. The antibody componentcan bind to PD-1, PD-L1, and/or a PD-L2 polypeptide. In one embodiment,the bispecific antibody could specifically bind to both a PD-1 ligandand a PD-1 polypeptide.

Yet another aspect of the invention pertains to anti-PD-1, PD-L1, orPD-L2 polypeptide antibodies that are obtainable by a processcomprising, immunizing an animal with an immunogenic PD-1, PD-L1, orPD-L2 polypeptide, respectively, or an immunogenic portion thereof; andthen isolating from the animal antibodies that specifically bind to thepolypeptide.

In still another aspect of the invention, partial or known antibodysequences can be used to generate and/or express new antibodies.Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L et al., 1998, Nature332:323 327; Jones, P. et al., 1986, Nature 321:522 525; and Queen, C.et al., 1989, Proc. Natl. Acad. See. U.S.A. 86:10029 10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline or non-germline antibody gene sequences. These germlinesequences will differ from mature antibody gene sequences because theywill not include completely assembled variable genes, which are formedby V(D)J joining during B cell maturation. Germline gene sequences willalso differ from the sequences of a high affinity secondary repertoireantibody at individual evenly across the variable region. For example,somatic mutations are relatively infrequent in the amino-terminalportion of framework region. For example, somatic mutations arerelatively infrequent in the amino terminal portion of framework region1 and in the carboxy-terminal portion of framework region 4.Furthermore, many somatic mutations do not significantly alter thebinding properties of the antibody. For this reason, it is not necessaryto obtain the entire DNA sequence of a particular antibody in order torecreate an intact recombinant antibody having binding propertiessimilar to those of the original antibody (see PCT/US99/05535 filed onMar. 12, 1999). Partial heavy and light chain sequence spanning the CDRregions is typically sufficient for this purpose. The partial sequenceis used to determine which germline and/or non-germline variable andjoining gene segments contributed to the recombined antibody variablegenes. The germline and/or non-germline sequence is then used to fill inmissing portions of the variable regions. Heavy and light chain leadersequences are cleaved during protein maturation and do not contribute tothe properties of the final antibody. To add missing sequences, clonedcDNA sequences can be combined with synthetic oligonucleotides byligation or PCR amplification. Alternatively, the entire variable regioncan be synthesized as a set of short, overlapping, oligonucleotides andcombined by PCR amplification to create an entirely synthetic variableregion clone. This process has certain advantages such as elimination orinclusion or particular restriction sites, or optimization of particularcodons. The process can also be used to screen libraries of particularimmunoglobulin encoding sequences in one species (e.g., human) to designcognate immunoglobulin encoding sequences from known antibody sequencein another species (e.g., mouse) (see, for example, the Examples sectionbelow).

The nucleotide sequences of heavy and light chain transcripts from ahybridoma are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak,1991, J. Biol. Chem. 266L19867019870); and, HindIII sites are engineeredupstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding, and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotide approximately the midpoint of the correspondingnon-coding oligonucleotide. Thus, for each chain, the oligonucleotidescan be assembled into overlapping double stranded sets that spansegments of 150-400 nucleotides. The pools are then used as templates toproduce PCR amplification products of 150-400 nucleotides. Typically, asingle variable region oligonucleotide set will be broken down into twopools which are separately amplified to generate two overlapping PCRproducts. These overlapping products are then combined by PCRamplification to form the complete variable region. It may also bedesirable to include an overlapping fragment of the heavy or light chainconstant region in the PCR amplification to generate fragments that caneasily be cloned into the expression vector constructs.

The reconstructed heavy and light chain variable regions are thencombined with cloned promoter, leader sequence, translation initiation,leader sequence, constant region, 3′ untranslated, polyadenylation, andtranscription termination, sequences to form expression vectorconstructs. The heavy and light chain expression constructs can becombined into a single vector, co-transfected, serially transfected, orseparately transfected into host cells which are then fused to form ahost cell expressing both chains.

Plasmids for this use are known in the art and include the plasmidsprovided in the Examples section below. Fully human and chimericantibodies of the present invention also include IgG2, IgG3, IgE, IgA,IgM, and IgD antibodies. Similar plasmids can be constructed forexpression of other heavy chain isotypes, or for expression ofantibodies comprising lambda light chains.

Thus, in another aspect of the invention, the structural features ofknown, non-human or human antibodies (e.g., a mouse anti-humananti-PD-1, PD-L1, or PD-L2 antibody, such as antibodies EH12.2H7,29E.2A3, and 24F.10C12 respectively) are used to create structurallyrelated human anti-human PD-1, PD-L1, or PD-L2 antibodies that retain atleast one functional property of the antibodies of the invention, suchas binding to PD-1, PD-L1, or PD-L2. Another functional propertyincludes inhibiting binding of EH12.2H7 to PD-1, 29E.2A3 to PD-L1, or24F.10C12 to PD-L2 in a competition ELISA assay. In some embodiments,the structurally related anti-human PD-1, PD-L1, or PD-L2 antibodieshave a lower binding affinity to the antigen as compared to antibodyEH12.2H7, 29E.2A3, or 24F.10C12 as measured by the IC50 value asdescribed in Example 2 (e.g., the affinity of the murine referenceantibody is no greater than any of 3.0, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5,1.4, 1.3, 1.2 or 1.1 fold of the structurally related antibody). In someembodiments, the structurally related anti-human PD-1, PD-L1, or PD-L2antibodies have a higher affinity to the antigen as compared to antibodyEH12.2H7, 29E.2A3, or 24F.10C12 as measured by the IC50 value asdescribed in Example 2 (such as the affinity of the structurally relatedantibody is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0fold of the reference antibody). In addition, one or more CDR orvariable regions of the present invention (e.g., FIGS. 2-7) can becombined recombinantly with known human framework regions and CDRs tocreate additional, recombinantly-engineered, human anti-PD-1, PD-L1, orPD-L2 antibodies of the invention.

Since it is well known in the art that antibody heavy and light chainCDR3 domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen, the recombinantantibodies of the invention prepared as set forth above preferablycomprise the heavy and light chain CDR3s of variable regions of thepresent invention (e.g., FIGS. 2-7). The antibodies further can comprisethe CDR2s of variable regions of the present invention (e.g., FIGS.2-7). The antibodies further can comprise the CDR1s of variable regionsof the present invention (e.g., FIGS. 2-7). The antibodies can furthercomprise any combinations of the CDRs.

The CDR1, 2, and/or 3 regions of the engineered antibodies describedabove can comprise the exact amino acid sequence(s) as those of variableregions of the present invention (e.g., FIGS. 2-7) disclosed herein.However, the ordinarily skilled artisan will appreciate that somedeviation from the exact CDR sequences may be possible while stillretaining the ability of the antibody to bind PD-1, PD-L1, or PD-L2effectively (e.g., conservative sequence modifications). Accordingly, inanother embodiment, the engineered antibody may be composed of one ormore CDRs that are, for example, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to one or moreCDRs of the present invention (e.g., FIGS. 2-7).

In addition to simply binding PD-1, PD-L1, or PD-L2, engineeredantibodies such as those described above may be selected for theirretention of other functional properties of antibodies of the invention,such as:

(1) binding to human PD-1, PD-L1, or PD-L2;(2) inhibiting binding of EH12.2H7 to PD-1, 29E.2A3 to PD-L1, or24F.10C12 to PD-L2;(3) binding to human PD-1 and inhibiting the ability of the bound PD-1to bind to PD-1 ligands (e.g., PD-L1 and/or PD-L2);(4) binding to human PD-L1 and inhibiting the ability of the bound PD-L1to bind to PD-L1 ligands (e.g., PD-1 and/or B7-1);(5) binding to human PD-L2 and inhibiting the ability of the bound PD-L2to bind to PD-L2 ligands (e.g., PD-1).

Heavy and light chain variable region amino acid sequences for antibodyEH12.2H7, 29E.2A3 and 24F.10C12 are shown below.

EH12.2H7 heavy chain variable region (SEQ ID NO: 76)QVQLQQSGAELAKPGASVQMSCKASGYSFTSSWIHWVKQRPGQGLEWIGYIYPSTGFTEYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWRDSSGYHAMDYWGQGTSVTVSS  EH12.2H7 light chain variable region(SEQ ID NO: 77) DIVLTQSPASLTVSLGQRATISCRASQSVSTSGYSYMHWYQQKPGQPPKLLIKFGSNLESGIPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEI PYTFGGGTKLEIK 29E.2A3 heavy chain variable region (SEQ ID NO: 78)EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAR QAWGYPWGQGTLVTVSA 29E.2A3 light chain variable region (SEQ ID NO: 79)DIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPY TFGGGTKLEIK 24F.10C12 heavy chain variable region (SEQ ID NO: 80)QVQLQQSAAELARPGASVKMSCKASGYTFTGYTMHWVKQRPGQGLEWIGYINPRSGYTEYNQKFKDKTTLTADKSSSTAYMQLSSLTSEDSAVYYCARPW FAYWGQGTLVTVSA24F.10C12 light chain variable region (SEQ ID NO: 81)DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSY PLTFGAGTKLELK 

Antibodies' activity in inhibiting binding of PD-1, PD-L1, or PD-L2 toits ligand(s) can be determined by testing the ability of the antibodyfrom blocking the binding between PD-1, PD-L1, or PD-L2 and its ligand.A competition ELISA assay in the presence of a labeled ligand and theantibody may be used. For example, to determine if an anti-PD-L1antibody could block the interaction between PD-1 and PD-L1, acompetitive binding experiment is performed. Cells expressing PD-L1 ispreincubated with the anti-PD-L1 antibody followed by the addition ofbiotinylated PD-1-Ig fusion protein. If the anti-PD-L1 antibody blocksthe binding of PD-1-Ig in a dose-dependent manner and with high avidity,the antib-PD-L1 antibody is considered as being effective in inhibitingthe interaction between PD-1 and PD-L1. Similar tests may be carried outto test antibodies that are effective in inhibiting the interaction ofPD-1 and PD-L2.

IV. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing one, two, or more nucleic acid moleculesencoding one or more polypeptides of the present invention (e.g., FIGS.2-7) (or a portion thereof). As used herein, the term “vector” refers toa nucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel (1990) Methods Enzymol. 185:3-7.Regulatory sequences include those which direct constitutive expressionof a nucleotide sequence in many types of host cells and those whichdirect expression of the nucleotide sequence only in certain host cells(e.g., tissue-specific regulatory sequences). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein.

The recombinant expression vectors of the invention can be designed forexpression of polypeptides of the present invention (e.g., FIGS. 2-7) inprokaryotic or eukaryotic cells. For example, the polypeptides can beexpressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel (1990) supra.Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of polypeptides in prokaryotes is most often carried out inE. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a polypeptide encoded therein,usually to the amino terminus of the recombinant polypeptide. Suchfusion vectors typically serve three purposes: 1) to increase expressionof recombinant polypeptide; 2) to increase the solubility of therecombinant polypeptide; and 3) to aid in the purification of therecombinant polypeptide by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantpolypeptide to enable separation of the recombinant polypeptide from thefusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to thetarget recombinant polypeptide.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al. (1988) Gene 69:301-315) and pET 1 Id (Studieret al. (1990) Methods Enzymol. 185:60-89). Target gene expression fromthe pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11 dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gnl). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident prophage harboring a T7 gnl gene under the transcriptionalcontrol of the lacUV 5 promoter.

One strategy to maximize recombinant polypeptide expression in E. coliis to express the polypeptide in host bacteria with impaired capacity toproteolytically cleave the recombinant polypeptide (Gottesman, S. (1990)Methods Enzymol. 185:119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjanand Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987)Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, polypeptides of the present invention (e.g., FIGS. 2-7)can be expressed in insect cells using baculovirus expression vectors.Baculovirus vectors available for expression of polypeptides in culturedinsect cells (e.g., Sf 9 cells) include the pAc ‘series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the present invention(e.g., FIGS. 2-7) is expressed in mammalian cells using a mammalianexpression vector. Examples of mammalian expression vectors includepCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)EMBO J. 6:187-195). When used in mammalian cells, the expressionvector's control functions are often provided by viral regulatoryelements. For example, commonly used promoters are derived from polyoma,Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitableexpression systems for both prokaryotic and eukaryotic cells seechapters 16 and 17 of Sambrook, J. et al., Molecular Cloning: ALaboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N. Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example by the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the .alpha.-fetoprotein promoter (Campesand Tilghman (1989) Genes Dev. 3:537-546).

Another aspect of the invention pertains to host cells into which anucleic acid molecule of the present invention (e.g., FIGS. 2-7) isintroduced within a recombinant expression vector or a nucleic acidmolecule containing sequences which allow it to homologously recombineinto a specific site of the host cell's genome. The terms “host cell”and “recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, apolypeptide of the present invention (e.g., FIGS. 2-7) can be expressedin bacterial cells such as E. coli, insect cells, yeast or mammaliancells (such as Chinese hamster ovary cells (CHO) or COS cells). Othersuitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a PD-L2 polypeptide or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a polypeptide ofthe present invention (e.g., FIGS. 2-7). Accordingly, the inventionfurther provides methods for producing a polypeptide of the presentinvention (e.g., FIGS. 2-7) using the host cells of the presentinvention. In one embodiment, the method comprises culturing the hostcell of the invention (into which a recombinant expression vectorencoding a polypeptide of the present invention (e.g., FIGS. 2-7) hasbeen introduced) in a suitable medium such that a polypeptide of thepresent invention (e.g., FIGS. 2-7) is produced. In another embodiment,the method further comprises isolating a polypeptide of the presentinvention (e.g., FIGS. 2-7) from the medium or the host cell.

The host cells of the invention can also be used to produce non-humantransgenic animals, as described below.

V. Production of Transgenic and Transchromosomal Nonhuman Animals whichGenerate Composite, Human PD-1, PD-L1, or PD-L2 Antibodies

In yet another aspect, the invention provides transgenic andtranschromosomal non-human animals, such as transgenic ortranschromosomal mice, which are capable of expressing human monoclonalantibodies that specifically bind to PD-1, PD-L1, or PD-L2. In aparticular embodiment, the invention provides a transgenic ortranschromosomal mouse having a genome comprising a human heavy chaintransgene, such that the mouse produces human anti-PD-1, PD-L1, or PD-L2antibodies when immunized with PD-1, PD-L1, or PD-L2 antigen and/orcells expressing PD-1, PD-L1, or PD-L2. The human heavy chain transgenecan be integrated into the chromosomal DNA of the mouse, as is the casefor transgenic, e.g., HuMAb, mice accordingly to methods well known inthe art. Alternatively, the human heavy chain transgene can bemaintained extrachromosomally, as is the case for transchromosomal(e.g., KM) mice as described in WO 02/43478. Such transgenic andtranschromosomal mice are capable of producing multiple isotypes ofhuman monoclonal antibodies to PD-1, PD-L1, or PD-L2 (e.g., IgG, IgAand/or IgE) by undergoing V-D-J recombination and isotype switching.Isotype switching may occur by, e.g., classical or non-classical isotypeswitching.

The design of a transgenic or transchromsomal non-human animal thatresponds to foreign antigen stimulation with a heterologous antibodyrepertoire, requires that the heterologous immunoglobulin transgenescontained within the transgenic animal function correctly throughout thepathway of B-cell development. This includes, for example, isotypeswitching of the heterologous heavy chain transgene. Accordingly,transgenes are constructed so as to produce isotype switching and one ormore of the following of antibodies: (1) high level and cell-typespecific expression, (2) functional gene rearrangement, (3) activationof and response to allelic exclusion, (4) expression of a sufficientprimary repertoire, (5) signal transduction, (6) somatic hypermutation,and (7) domination of the transgene antibody locus during the immuneresponse.

Not all of the foregoing criteria need be met. For example, in thoseembodiments wherein the endogenous immunoglobulin loci of the transgenicanimal are functionally disrupted, the transgene need not activateallelic exclusion. Further, in those embodiments wherein the transgenecomprises a functionally rearranged heavy and/or light chainimmunoglobulin gene, the second criteria of functional generearrangement is unnecessary, at least for that transgene which isalready rearranged. For background on molecular immunology, see,Fundamental Immunology, 2nd edition (1989), Paul William E., ed. RavenPress, N.Y.

In certain embodiments, the transgenic or transchromosomal non-humananimals used to generate the human monoclonal antibodies of theinvention contain rearranged, unrearranged or a combination ofrearranged and unrearranged heterologous immunoglobulin heavy and lightchain transgenes in the germline of the transgenic animal. Each of theheavy chain transgenes comprises at least one CH gene. In addition, theheavy chain transgene may contain functional isotype switch sequences,which are capable of supporting isotype switching of a heterologoustransgene encoding multiple CH genes in the B-cells of the transgenicanimal. Such switch sequences may be those which occur naturally in thegermline immunoglobulin locus from the species that serves as the sourceof the transgene CH genes, or such switch sequences may be derived fromthose which occur in the species that is to receive the transgeneconstruct (the transgenic animal). For example, a human transgeneconstruct that is used to produce a transgenic mouse may produce ahigher frequency of isotype switching events if it incorporates switchsequences similar to those that occur naturally in the mouse heavy chainlocus, as presumably the mouse switch sequences are optimized tofunction with the mouse switch recombinase enzyme system, whereas thehuman switch sequences are not. Switch sequences may be isolated andcloned by conventional cloning methods, or may be synthesized de novofrom overlapping synthetic oligonucleotides designed on the basis ofpublished sequence information relating to immunoglobulin switch regionsequences (Mills et al., Nucl. Acids Res. 15:7305 7316 (1991); Sideraset al., Intl. Immunol. 1:631 642 (1989)). For each of the foregoingtransgenic animals, functionally rearranged heterologous heavy and lightchain immunoglobulin transgenes are found in a significant fraction ofthe B-cells of the transgenic animal (at least 10 percent).

The transgenes used to generate the transgenic animals of the inventioninclude a heavy chain transgene comprising DNA encoding at least onevariable gene segment, one diversity gene segment, one joining genesegment and at least one constant region gene segment. Theimmunoglobulin light chain transgene comprises DNA encoding at least onevariable gene segment, one joining gene segment and at least oneconstant region gene segment. The gene segments encoding the light andheavy chain gene segments are heterologous to the transgenic non-humananimal in that they are derived from, or correspond to, DNA encodingimmunoglobulin heavy and light chain gene segments from a species notconsisting of the transgenic non-human animal. In one aspect of theinvention, the transgene is constructed such that the individual genesegments are unrearranged, i.e., not rearranged so as to encode afunctional immunoglobulin light or heavy chain. Such unrearrangedtransgenes support recombination of the V, D, and J gene segments(functional rearrangement) and preferably support incorporation of allor a portion of a D region gene segment in the resultant rearrangedimmunoglobulin heavy chain within the transgenic non-human animal whenexposed to the PD-1, PD-L1, or PD-L2 antigen.

In an alternate embodiment, the transgenes comprise an unrearranged“mini-locus”. Such transgenes typically comprise a substantial portionof the C, D, and J segments as well as a subset of the V gene segments.In such transgene constructs, the various regulatory sequences, e.g.,promoters, enhancers, class switch regions, splice-donor andsplice-acceptor sequences for RNA processing, recombination signals andthe like, comprise corresponding sequences derived from the heterologousDNA. Such regulatory sequences may be incorporated into the transgenefrom the same or a related species of the non-human animal used in theinvention. For example, human immunoglobulin gene segments may becombined in a transgene with a rodent immunoglobulin enhancer sequencefor use in a transgenic mouse. Alternatively, synthetic regulatorysequences may be incorporated into the transgene, wherein such syntheticregulatory sequences are not homologous to a functional DNA sequencethat is known to occur naturally in the genomes of mammals. Syntheticregulatory sequences are designed according to consensus rules, such as,for example, those specifying the permissible sequences of asplice-acceptor site or a promoter/enhancer motif. For example, aminilocus comprises a portion of the genomic immunoglobulin locus havingat least one internal (i.e., not at a terminus of the portion) deletionof a non-essential DNA portion (e.g., intervening sequence; intron orportion thereof) as compared to the naturally-occurring germline Iglocus.

Transgenic and transchromsomal mice employed in the present inventioncan exhibit immunoglobulin production with a significant repertoire,ideally substantially similar to that of a native mouse. Thus, forexample, in embodiments where the endogenous Ig genes have beeninactivated, the total immunoglobulin levels can range from about 0.1 to10 mg/ml of serum, or from about 0.5 to 5 mg/ml, or at least about 1.0mg/ml. When a transgene capable of effecting a switch to IgG from IgMhas been introduced into the transgenic mouse, the adult mouse ratio ofserum IgG to IgM can be about 10:1. The IgG to IgM ratio will be muchlower in the immature mouse. In general, greater than about 10%,preferably 40 to 80% of the spleen and lymph node B cells expressexclusively human IgG protein.

The repertoire will ideally approximate that shown in a native mouse,usually at least about 10% as high, or 25 to 50% or more. Generally, atleast about a thousand different immunoglobulins (ideally IgG), e.g.,preferably 10⁴ to 10⁶ or more, will be produced, depending primarily onthe number of different V, J and D regions introduced into the mousegenome. These immunoglobulins will typically recognize about one-half ormore of highly antigenic proteins, e.g., staphylococcus protein A.Typically, the immunoglobulins will exhibit an affinity (K_(D)) forpreselected antigens of below 10⁻⁷ M, such as of below 10⁻⁸ M, 10⁻⁹ M or10⁻¹⁰ M or even lower.

In some embodiments, it may be preferable to generate mice withpredetermined repertoires to limit the selection of V genes representedin the antibody response to a predetermined antigen type. A heavy chaintransgene having a predetermined repertoire may comprise, for example,human V_(H) genes which are preferentially used in antibody responses tothe predetermined antigen type in humans. Alternatively, some V_(H)genes may be excluded from a defined repertoire for various reasons(e.g., have a low likelihood of encoding high affinity V regions for thepredetermined antigen; have a low propensity to undergo somatic mutationand affinity sharpening; or are immunogenic to certain humans) Thus,prior to rearrangement of a transgene containing various heavy or lightchain gene segments, such gene segments may be readily identified, e.g.by hybridization or DNA sequencing, as being from a species of organismother than the transgenic animal.

Transgenic and transchromosomal mice as described above can be immunizedwith, for example, a purified or enriched preparation of PD-1, PD-L1, orPD-L2 antigen and/or cells expressing PD-1, PD-L1, or PD-L2.Alternatively, the transgenic mice can be immunized with DNA encodinghuman PD-1, PD-L1, or PD-L2. The mice will then produce B cells whichundergo class-switching via intratransgene switch recombination(cis-switching) and express immunoglobulins reactive with PD-1, PD-L1,or PD-L2. The immunoglobulins can be human antibodies (also referred toas “human sequence antibodies”), wherein the heavy and light chainpolypeptides are encoded by human transgene sequences, which may includesequences derived by somatic mutation and V region recombinatorialjoints, as well as germline-encoded sequences; these human antibodiescan be referred to as being substantially identical to a polypeptidesequence encoded by a human V_(L) or V_(H) gene segment and a humanJ_(L) or D_(H) and J_(H) segment, even though other non-germlinesequences may be present as a result of somatic mutation anddifferential V-J and V-D-J recombination joints. The variable regions ofeach antibody chain are typically at least 80 percent encoded by humangermline V, J, and, in the case of heavy chains, D, gene segments;frequently at least 85 percent of the variable regions are encoded byhuman germline sequences present on the transgene; often 90 or 95percent or more of the variable region sequences are encoded by humangermline sequences present on the transgene. However, since non-germlinesequences are introduced by somatic mutation and VJ and VDJ joining, thehuman sequence antibodies will frequently have some variable regionsequences (and less frequently constant region sequences) which are notencoded by human V, D, or J gene segments as found in the humantransgene(s) in the germline of the mice. Typically, such non-germlinesequences (or individual nucleotide positions) will cluster in or nearCDRs, or in regions where somatic mutations are known to cluster.

Human antibodies which bind to the predetermined antigen can result fromisotype switching, such that human antibodies comprising a humansequence γ chain (such as γ1, γ 2a, γ 2B, or γ 3) and a human sequencelight chain (such as kappa) are produced. Such isotype-switched humanantibodies often contain one or more somatic mutation(s), typically inthe variable region and often in or within about 10 residues of a CDR)as a result of affinity maturation and selection of B cells by antigen,particularly subsequent to secondary (or subsequent) antigen challenge.These high affinity human antibodies may have binding affinities (K_(D))of below 10⁻⁷ M, such as of below 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹⁰ M oreven lower.

Another aspect of the invention includes B cells derived from transgenicor transchromosomal mice as described herein. The B cells can be used togenerate hybridomas expressing human monoclonal antibodies which bindwith high affinity (e.g., lower than 10⁻⁷ M) to human PD-1, PD-L1, orPD-L2.

The development of high affinity human monoclonal antibodies againstPD-1, PD-L1, or PD-L2 can be facilitated by a method for expanding therepertoire of human variable region gene segments in a transgenic mousehaving a genome comprising an integrated human immunoglobulin transgene,said method comprising introducing into the genome a V gene transgenecomprising V region gene segments which are not present in saidintegrated human immunoglobulin transgene. Often, the V region transgeneis a yeast artificial chromosome comprising a portion of a human V_(H)or V_(L) (V_(K)) gene segment array, as may naturally occur in a humangenome or as may be spliced together separately by recombinant methods,which may include out-of-order or omitted V gene segments. Often atleast five or more functional V gene segments are contained on the YAC.In this variation, it is possible to make a transgenic mouse produced bythe V repertoire expansion method, wherein the mouse expresses animmunoglobulin chain comprising a variable region sequence encoded by aV region gene segment present on the V region transgene and a C regionencoded on the human Ig transgene. By means of the V repertoireexpansion method, transgenic mice having at least 5 distinct V genes canbe generated; as can mice containing at least about 24 V genes or more.Some V gene segments may be non-functional (e.g., pseudogenes and thelike); these segments may be retained or may be selectively deleted byrecombinant methods available to the skilled artisan, if desired.

Once the mouse germline has been engineered to contain a functional YAChaving an expanded V segment repertoire, substantially not present inthe human Ig transgene containing the J and C gene segments, the traitcan be propagated and bred into other genetic backgrounds, includingbackgrounds where the functional YAC having an expanded V segmentrepertoire is bred into a mouse germline having a different human Igtransgene. Multiple functional YACs having an expanded V segmentrepertoire may be bred into a germline to work with a human Ig transgene(or multiple human Ig transgenes). Although referred to herein as YACtransgenes, such transgenes when integrated into the genome maysubstantially lack yeast sequences, such as sequences required forautonomous replication in yeast; such sequences may optionally beremoved by genetic engineering (e.g., restriction digestion andpulsed-field gel electrophoresis or other suitable method) afterreplication in yeast is no longer necessary (i.e., prior to introductioninto a mouse ES cell or mouse prozygote). Methods of propagating thetrait of human sequence immunoglobulin expression, include breeding atransgenic mouse having the human Ig transgene(s), and optionally alsohaving a functional YAC having an expanded V segment repertoire. BothV_(H) and V_(L) gene segments may be present on the YAC. The transgenicmouse may be bred into any background desired by the practitioner,including backgrounds harboring other human transgenes, including humanIg transgenes and/or transgenes encoding other human lymphocyteproteins. The invention also provides a high affinity human sequenceimmunoglobulin produced by a transgenic mouse having an expanded Vregion repertoire YAC transgene. Although the foregoing describes apreferred embodiment of the transgenic animal of the invention, otherembodiments are contemplated which have been classified in fourcategories:

(1) Transgenic animals containing an unrearranged heavy and rearrangedlight immunoglobulin transgene;(2) Transgenic animals containing an unrearranged heavy and unrearrangedlight immunoglobulin transgene;(3) Transgenic animal containing rearranged heavy and an unrearrangedlight immunoglobulin transgene; and(4) Transgenic animals containing rearranged heavy and rearranged lightimmunoglobulin transgenes.

VI. Antibody Conjugates/Immunotoxins

In another aspect, the present invention features human PD-1, PD-L1, orPD-L2 antibodies conjugated to a therapeutic moiety, such as acytotoxin, a drug (e.g., an immunosuppressant) or a radioisotope. Whenconjugated to a cytotoxin, these antibody conjugates are referred to as“immunotoxins.” A cytotoxin or cytotoxic agent includes any agent thatis detrimental to (e.g., kills) cells. Examples include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). An antibody of the presentinvention can be conjugated to a radioisotope, e.g., radioactive iodine,to generate cytotoxic radiopharmaceuticals for treating a relateddisorder, such as a cancer.

Conjugated human PD-1, PD-L1, or PD-L2 antibodies can be useddiagnostically or prognostically to monitor polypeptide levels in tissueas part of a clinical testing procedure, e.g., to, for example,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling (i e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, P-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ₁₂₅I, ₁₃₁I, ₃₅S or₃H.

The antibody conjugates of the invention can be used to modify a givenbiological response. The therapeutic moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-.gamma.; or, biological response modifierssuch as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other cytokines or growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243 56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623 53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303 16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119 58 (1982).

VII. Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination of themonoclonal antibodies, or antigen-binding portion(s) thereof (such asantigen-binding fragments), of the present invention, formulatedtogether with a pharmaceutically acceptable carrier. In one embodiment,the compositions include a combination of multiple (e.g., two or more)isolated human antibodies of the invention. Preferably, each of theantibodies of the composition binds to a distinct, pre-selected epitopeof PD-1, PD-L1, and/or PD-L2.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a composition of the present inventionwith at least one or more additional therapeutic agents, such asanti-inflammatory agents, DMARDs (disease-modifying anti-rheumaticdrugs), immunosuppressive agents, chemotherapeutics, and psoriasisagents. The pharmaceutical compositions of the invention can also beadministered in conjunction with radiation therapy. Co-administrationwith other antibodies, such as CD4 specific antibodies and IL-2 specificantibodies, are also encompassed by the invention.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,bispecific and multispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1 19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

To administer a compound of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. For example, the humanantibodies of the invention may be administered once or twice weekly bysubcutaneous injection or once or twice monthly by subcutaneousinjection. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

In one embodiment, an agent of the invention is an antibody. As definedherein, a therapeutically effective amount of antibody (i.e., aneffective dosage) ranges from about 0.001 to 30 mg/kg body weight, orabout 0.01 to 25 mg/kg body weight, or about 0.1 to 20 mg/kg bodyweight, or about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciatethat certain factors may influence the dosage required to effectivelytreat a subject, including but not limited to the severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject, and other diseases present. Moreover, treatment of asubject with a therapeutically effective amount of an antibody caninclude a single treatment or, preferably, can include a series oftreatments. It will also be appreciated that the effective dosage ofantibody used for treatment may increase or decrease over the course ofa particular treatment. Changes in dosage may result from the results ofdiagnostic assays.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present inventioninclude those suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect. Generally, out of one hundredpercent, this amount will range from about 0.001 percent to about ninetypercent of active ingredient, alternatively from about 0.005 percent toabout 70 percent, or alternatively from about 0.01 percent to about 30percent.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate. Dosage forms for the topical or transdermaladministration of compositions of this invention include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrastemal injection andinfusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.001 to 90% (e.g.,0.005 to 70%, such as 0.01 to 30%) of active ingredient in combinationwith a pharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. A physician orveterinarian having ordinary skill in the art can readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician or veterinarian could start dosesof the compounds of the invention employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. In general, a suitable daily dose of acompositions of the invention will be that amount of the compound whichis the lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described above.It is preferred that administration be intravenous, intramuscular,intraperitoneal, or subcutaneous, preferably administered proximal tothe site of the target. If desired, the effective daily dose of atherapeutic composition may be administered as two, three, four, five,six or more sub-doses administered separately at appropriate intervalsthroughout the day, optionally, in unit dosage forms. While it ispossible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation (composition).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in one embodiment, a therapeutic composition ofthe invention can be administered with a needleless hypodermic injectiondevice, such as the devices disclosed in U.S. Pat. Nos. 5,399,163,5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.Examples of well-known implants and modules useful in the presentinvention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many othersuch implants, delivery systems, and modules are known to those skilledin the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134),different species of which may comprise the formulations of theinventions, as well as components of the invented molecules; p120(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273. In one embodiment of the invention, thetherapeutic compounds of the invention are formulated in liposomes; inanother embodiment, the liposomes include a targeting moiety. In yetanother embodiment, the therapeutic compounds in the liposomes aredelivered by bolus injection to a site proximal to the tumor orinfection. The composition must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound may be orally administered, for example, with an inert diluentor an assimilable edible carrier.

VIII. Uses and Methods of the Invention

The antibodies described herein (including derivatives and conjugates ofthe antibodies) and compositions containing the antibodies can be usedin a variety of in vitro and in vivo diagnostic and therapeuticapplications (e.g., by up- or down-modulating the immune response). Forexample, PD-1 ligand binding to PD-1 or B7-1 transmits an inhibitorysignal. Thus, modulation of the interaction between PD-1 and a PD-1ligand, or between a PD-1 ligand and a B7 polypeptide, results inmodulation of the immune response. PD-1 ligands can also costimulate Tcells. Thus, in one embodiment, antibodies which block the interactionbetween a PD-1 ligand and PD-1 or B7 can prevent inhibitory signaling.In one embodiment, antibodies that block costimulatory signal of thePD-1 ligand block a costimulatory signal to an immune cell. Furthermore,ligation of PD-L2 can induce cytokine secretion and survival ofdendritic cells. Thus, antibodies that block PD-L2 ligation can inhibitdendritic cell survival and reduce cytokine expression by dendriticcells, and through these mechanisms inhibit an immune response. Inparticular, antibodies described herein are useful for diagnostic,prognostic, prevention, and therapeutic applications related toparticular conditions mediated by PD-1, PD-L1, and/or PD-L2, asdiscussed, for example, in Keir et al. (2008) Annu. Rev. Immunol.26:677; Sharpe et al., (2007) Nat. Immunol. 8:239; Freeman et al. (2007)J. Exp. Med. 10:2223; each of which is hereby incorporated by referencein their entirety.

In one embodiment, the antibodies and the antigen-binding fragments ofthe present invention are useful for diagnostic, prognostic, prevention,and therapeutic applications regarding neurodegenerative diseases(geriopsychosis, Alzheimer disease, Down syndrome, Parkinson's disease,Creutzfeldt-jakob disease, diabetic neuropathy, Parkinson syndrome,Huntington's disease, Machado-Joseph disease, amyotrophic lateralsclerosis, diabetic neuropathy, and Creutzfeldt Creutzfeldt-Jakobdisease).

In another embodiment, the antibodies and the antigen-binding fragmentsof the present invention are useful diagnostic, prognostic, prevention,and therapeutic applications (such as treating, and delaying the onsetor progression of the diseases) for diseases that accelerate the immunereaction, for example, asthma, autoimmune diseases (glomerularnephritis, arthritis, dilated cardiomyopathy-like disease, ulceouscolitis, Sjogren syndrome, Crohn disease, systemic erythematodes,chronic rheumatoid arthritis, multiple sclerosis, psoriasis, allergiccontact dermatitis, polymyosiis, pachyderma, periarteritis nodosa,rheumatic fever, vitiligo vulgaris, insulin dependent diabetes mellitus,Behcet disease, Hashimoto disease, Addison disease, dermatomyositis,myasthenia gravis, Reiter syndrome, Graves' disease, anaemia perniciosa,Goodpasture syndrome, sterility disease, chronic active hepatitis,pemphigus, autoimmune thrombopenic purpura, and autoimmune hemolyticanemia, active chronic hepatitis, Addison's disease, anti-phospholipidsyndrome, atopic allergy, autoimmune atrophic gastritis, achlorhydraautoimmune, celiac disease, Cushing's syndrome, dermatomyositis, discoidlupus, erythematosis, Goodpasture's syndrome, Hashimoto's thyroiditis,idiopathic adrenal atrophy, idiopathic thrombocytopenia,insulin-dependent diabetes, Lambert-Eaton syndrome, lupoid hepatitis,some cases of lymphopenia, mixed connective tissue disease, pemphigoid,pemphigus vulgaris, pernicious anema, phacogenic uveitis, polyarteritisnodosa, polyglandular autosyndromes, primary biliary cirrhosis, primarysclerosing cholangitis, Raynaud's syndrome, relapsing polychondritis,Schmidt's syndrome, limited scleroderma (or crest syndrome), sympatheticophthalmia, systemic lupus erythematosis, Takayasu's arteritis, temporalarteritis, thyrotoxicosis, type b insulin resistance, ulcerative colitisand Wegener's granulomatosis).

In still another embodiment, the antibodies and the antigen-bindingfragments of the present invention are useful diagnostic, prognostic,prevention, and therapeutic applications (such as treating, and delayingthe onset or progression of the diseases) for therapy and/or preventionfor persistent infectious disease (e.g., viral infectious diseasesincluding HPV, HBV, hepatitis C Virus (HCV), retroviruses such as humanimmunodeficiency virus (HIV-1 and HIV-2), herpes viruses such as EpsteinBarr Virus (EBV), cytomegalovirus (CMV), HSV-1 and HSV-2, and influenzavirus. Other antigens associated with pathogens that can be utilized asdescribed herein are antigens of various parasites, includes malaria,preferably malaria peptide based on repeats of NANP. In addition,bacterial, fungal and other pathogenic diseases are included, such asAspergillus, Brugia, Candida, Chlamydia, Coccidia, Cryptococcus,Dirofilaria, Gonococcus, Histoplasma, Leishmania, Mycobacterium,Mycoplasma, Paramecium, Pertussis, Plasmodium, Pneumococcus,Pneumocystis, Rickettsia, Salmonella, Shigella, Staphylococcus,Streptococcus, Toxoplasma and Vibriocholerae. Exemplary species includeNeisseria gonorrhea, Mycobacterium tuberculosis, Candida albicans,Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, GroupB Streptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granulomainguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus.Brucella melitensis, Brucella suis, Brucella canis, Campylobacter fetus,Campylobacter fetus intestinalis, Leptospira pomona, Listeriamonocytogenes, Brucella ovis, Chlamydia psittaci, Trichomonas foetus,Toxoplasma gondii, Escherichia coli, Actinobacillus equuli, Salmonellaabortus ovis, Salmonella abortus equi, Pseudomonas aeruginosa,Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis,Mycoplasma bovigenitalium, Aspergillus fumigatus, Absidia ramosa,Trypanosoma equiperdum, Babesia caballi, Clostridium tetani, Clostridiumbotulinum; or, a fungus, such as, e.g., Paracoccidioides brasiliensis;or other pathogen, e.g., Plasmodium falciparum. Also included areNational Institute of Allergy and Infectious Diseases (NIAID) prioritypathogens. These include Category A agents, such as variola major(smallpox), Bacillus anthracia (anthrax), Yersinia pestis (plague),Clostridium botulinum toxin (botulism), Francisella tularensis(tularaemia), filoviruses (Ebola hemorrhagic fever, Marburg hemorrhagicfever), arenaviruses (Lassa (Lassa fever), Junin (Argentine hemorrhagicfever) and related viruses); Category B agents, such as Coxiellaburnetti (Q fever), Brucella species (brucellosis), Burkholderia mallei(glanders), alphaviruses (Venezuelan encephalomyelitis, eastern &western equine encephalomyelitis), ricin toxin from Ricinus communis(castor beans), epsilon toxin of Clostridium perfringens; Staphylococcusenterotoxin B, Salmonella species, Shigella dysenteriae, Escherichiacoli strain O157:H7, Vibrio cholerae, Cryptosporidium parvum; Category Cagents, such as nipah virus, hantaviruses, tickborne hemorrhagic feverviruses, tickborne encephalitis viruses, yellow fever, andmultidrug-resistant tuberculosis; helminths, such as Schistosoma andTaenia; and protozoa, such as Leishmania (e.g., L. mexicana) andPlasmodium.

In yet another embodiment, the antibodies or the antigen-bindingfragments of the present invention are useful for diagnostic,prognostic, prevention, and therapeutic applications for organ graftrejection, graft-versus-host disease (GVHD), allergic disease, anddiseases caused by attenuation of immune reaction, which PD-1, PD-L1,and/or PD-L2 participates, for example, cancer and infectious disease.

The antibodies or antigen-binding fragments described herein areadministered to a subject in accord with known methods, such as byintravenous (e.g., as a bolus or by continuous infusion over a period oftime), subcutaneous, intramuscular, intraperitoneal, intracerobrospinal,intra-articular, intrasynovial, intrathecal, or inhalation routesadministration.

A subject is treated if one or more beneficial or desired results,including desirably clinical results, are obtained. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, one or more of the following: decreasing one or moresymptoms resulting from the disease, increasing the quality of life ofthose suffering from the disease, decreasing the dose of othermedications required to treat the disease, delaying the progression ofthe disease, and/or prolonging survival of individuals.

1. Screening Methods

One aspect of the present invention relates to methods of usingantibodies of the present to modulate an immune response by modulatingcostimulation (such as antibodies that modulate the function of PD-1,PD-L1, or PD-L2). Such methods utilize screening assays, including cellbased and non-cell based assays. In one embodiment, the assays provide amethod for identifying antibodies which modulate the interaction of aPD-1 ligand and PD-1. In another embodiment, the assays provide a methodfor identifying antibodies which modulate the interaction between a PD-1ligand and a B7 polypeptide.

In one embodiment, the invention relates to assays for screeningcandidate or test antibodies which bind to, or modulate the activity of,PD-1, PD-L1, or PD-L2, e.g., modulate the ability of the polypeptide tointeract with (e.g., bind to) its cognate binding partner. In oneembodiment, a method for identifying an antibody to modulate an immuneresponse entails determining the ability of the antibody to modulate, eg enhance or inhibit, the interaction between PD-1 and a PD-1 ligand,and further determining the ability of the antibody to modulate theinteraction between a PD-1 ligand and a B7 polypeptide. In oneembodiment, an antibody that modulates the interaction between the PD-1ligand and PD-1 (e.g., without modulating the interaction between thePD-1 ligand and the B7 polypeptide is selected). In another embodiment,an antibody that modulates the interaction between a PD-1 ligand and aB7 polypeptide (e.g., without modulating the interaction between thePD-1 ligand and PD-1) is selected.

In another embodiment, a method for identifying an antibody to decreasean immune response entails determining the ability of a candidateantibody to enhance the interaction between a PD-1 ligand and a B7polypeptide and selecting an antibody that inhibits the interactionbetween the PD-1 ligand and the B7 polypeptide. In another embodiment, amethod for identifying an antibody to decrease an immune responseentails determining the ability of the candidate antibody to enhance theinteraction between a PD-1 ligand and PD-1 and selecting an antibodythat enhances the interaction between the PD-1 ligand and PD-1

In one embodiment, an assay is a cell-based assay, comprising contactinga cell expressing PD-1, PD-L1, or PD-L2, with a test antibody anddetermining the ability of the test antibody to modulate (e.g. stimulateor inhibit) the binding of PD-1 or the PD-1 ligand target to its bindingpartner. Determining the ability of the PD-1, PD-1 ligand or B7polypeptide to bind to, or interact with, its binding partner can beaccomplished, e.g., by measuring direct binding or by measuring aparameter of immune cell activation.

For example, in a direct binding assay, the PD-1 or PD-1 ligand protein(or their respective target polypeptides) can be coupled with aradioisotope or enzymatic label such that binding of PD-1 ligand to PD-1or to the B7 polypeptide can be determined by detecting the labeledprotein in a complex. For example, PD-1 or PD-1 can be labeled with¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemmission or byscintillation counting. Alternatively, PD-1 or PD-1 ligand can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound to modulate the interaction between PD-1 and a PD-1 ligandor between a PD-1 ligand and a B7 polypeptide, without the labeling ofany of the interactants. For example, a microphysiometer can be used todetect the interaction of PD-1 and a PD-1 ligand, or between a PD-1ligand and a B7 polypeptide, with its target polypeptide, without thelabeling of either PD-1, PD-1 ligand, B7 polypeptide, or the targetpolypeptide (McConnell, H. M. et al. (1992) Science 257:1906-1912). Asused herein, a “microphysiometer” (e.g., Cytosensor) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between compound and receptor.

In another embodiment, determining the ability of the antibody toantagonize the interaction between a given set of polypeptides can beaccomplished by determining the activity of one or more members of theset of polypeptides. For example, the activity of PD-1 or a PD-1 ligandcan be determined by detecting induction of a cellular second messenger(e.g., tyrosine kinase activity), detecting catalytic/enzymatic activityof an appropriate substrate, detecting the induction of a reporter gene(comprising a target-responsive regulatory element operatively linked toa nucleic acid encoding a detectable marker, e.g., chloramphenicolacetyl transferase), or detecting a cellular response regulated by PD-1or the PD-1 ligand. Determining the ability of the antibody to bind toor interact with said polypeptide can be accomplished, for example, bymeasuring the ability of a compound to modulate immune cellcostimulation or inhibition in a proliferation assay, or by interferingwith the ability of said polypeptide to bind to antibodies thatrecognize a portion thereof.

Antibodies that block or inhibit interaction of a PD-1 ligand with acostimulatory receptor as well as antibodies that promote a PD-1ligand-mediated inhibitory signal can be identified by their ability toinhibit immune cell proliferation, and/or effector function, or toinduce anergy when added to an in vitro assay. For example, cells can becultured in the presence of an agent that stimulates signal transductionvia an activating receptor. A number of recognized readouts of cellactivation can be employed to measure, cell proliferation or effectorfunction (e.g., antibody production, cytokine production, phagocytosis)in the presence of the activating agent. The ability of a test antibodyto block this activation can be readily determined by measuring theability of the antibody to affect a decrease in proliferation oreffector function being measured, using techniques known in the art.

For example, antibodies of the present invention can be tested for theability to inhibit or enhance costimulation in a T cell assay, asdescribed in Freeman et al. (2000) J. Exp. Med. 192:1027 and Latchman etal. (2001) Nat. Immunol. 2:261. CD4+ T cells can be isolated from humanPBMCs and stimulated with activating anti-CD3 antibody. Proliferation ofT cells can be measured by ³H thymidine incorporation. An assay can beperformed with or without CD28 costimulation in the assay. Similarassays can be performed with Jurkat T cells and PHA-blasts from PBMCs.

In yet another embodiment, an assay of the present invention is acell-free assay in which PD-1 or a PD-1 ligand or a biologically activeportion thereof, is contacted with a test antibody, and the ability ofthe test antibody to bind to the polypeptide, or biologically activeportion thereof, is determined Binding of the test antibody to the PD-1or PD-1 ligand polypeptide can be determined either directly orindirectly as described above. In still another embodiment, the assayincludes contacting the polypeptide, or biologically active portionthereof, with its binding partner to form an assay mixture, contactingthe assay mixture with a test antibody, and determining the ability ofthe test antibody to interact with the polypeptide in the assay mixture,wherein determining the ability of the test antibody to interact withthe polypeptide comprises determining the ability of the test antibodyto preferentially bind to the polypeptide or biologically active portionthereof, as compared to the binding partner.

For example, a PD-1 ligand and a PD-1 polypeptide can be used to form anassay mixture and the ability of a test antibody to block thisinteraction can be tested by determining the ability of PD-1 to bind thePD-1 ligand and determining the ability of the PD-1 ligand to bind thePD-1 polypeptide, by one of the methods described above for determiningbinding. Determining the ability of a PD-1 polypeptide to bind a PD-1ligand and determining the ability of a PD-1 ligand to bind a B7polypeptide can also be accomplished using a technology such asreal-time Biomolecular Interaction Analysis (BIA) (Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” is atechnology for studying biospecific interactions in real time, withoutlabeling any of the interactants (e.g., BIAcore). Changes in the opticalphenomenon of surface plasmon resonance (SPR) can be used as anindication of real-time reactions between biological polypeptides. PD-1,PD-1 ligand, and B7 polypeptide can be immobilized on a BIAcore chip andantibodies can be tested for binding to PD-1, PD-1 ligand, and B7polypeptide. An example of using the BIA technology is described by Fitzet al. (1997) Oncogene 15:613.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of proteins (e.g., a PD-1ligand or PD-1 proteins or biologically active portions thereof, orbinding partners to which a PD-1 ligand or PD-1 binds). In the case ofcell-free assays in which a membrane-bound form protein is used (e.g., acell surface PD-1 ligand or PD-1 receptor) it may be desirable toutilize a solubilizing agent such that the membrane-bound form of theprotein is maintained in solution. Examples of such solubilizing agentsinclude non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In one or more embodiments of the above described assay methods, it maybe desirable to immobilize either PD-1, a PD-1 ligand, and a B7polypeptide, or an appropriate target polypeptide, to facilitateseparation of complexed from uncomplexed forms of one or both of theproteins, as well as to accommodate automation of the assay. Binding ofa test antibody to PD-1 or a PD-1 ligand can be accomplished in anyvessel suitable for containing the reactants. Examples of such vesselsinclude microtiter plates, test tubes, and micro-centrifuge tubes. Inone embodiment, a fusion protein can be provided which adds a domainthat allows one or both of the proteins to be bound to a matrix. Forexample, glutathione-S-transferase/PD-1, PD-1 ligand, or B7 polypeptidefusion proteins, or glutathione-S-transferase/target fusion proteins,can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.Louis, Mo.) or glutathione derivatized microtiter plates, which are thencombined with the test compound, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of PD-1,PD-1 ligand, or B7 polypeptide binding or activity determined usingstandard techniques.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of PD-1 or a PD-1 ligand can beaccomplished by determining the ability of the test antibody to modulatethe activity of a polypeptide that functions downstream of PD-1 or thePD-1 ligand, e.g., a polypeptide that interacts with the PD-1 ligand, ora polypeptide that functions downstream of PD-1, e.g., by interactingwith the cytoplasmic domain of PD-1. For example, levels of secondmessengers can be determined, the activity of the interactor polypeptideon an appropriate target can be determined, or the binding of theinteractor to an appropriate target can be determined as previouslydescribed.

This invention further pertains to novel antibodies identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an antibody identified as described hereinin an appropriate animal model. For example, an antibody identified asdescribed herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with such an antibody.Alternatively, an antibody identified as described herein can be used inan animal model to determine the mechanism of action of such anantibody. Furthermore, this invention pertains to uses of novelantibodies identified by the above-described screening assays fortreatments as described herein.

2. Prophylactic Methods

In one aspect, the invention relates to a method for preventing in asubject, a disease or condition associated with an unwanted or less thandesirable immune response. Subjects at risk for a disease that wouldbenefit from treatment with the claimed antibodies or methods can beidentified, for example, by any or a combination of diagnostic orprognostic assays known in the art. Administration of a prophylacticantibody can occur prior to the manifestation of symptoms associatedwith an unwanted or less than desirable immune response. The appropriateantibody used for treatment can be determined based on clinicalindications and can be identified, e.g., using screening assaysdescribed herein.

3. Therapeutic Methods

Another aspect of the invention pertains to therapeutic methods ofmodulating an immune response, e.g., by modulating the interactionbetween PD-1 and a PD-1 ligand and/or a PD-1 ligand and a B7polypeptide. For example, modulation of the interaction between PD-1 anda PD-1 ligand, or between a PD-1 ligand and a B7 polypeptide, results inmodulation of the immune response. Thus, in one embodiment, antibodieswhich block the interaction between PD-1 and the PD-1 ligand can preventinhibitory signaling. PD-1 ligands can also enhance costimulatorysignals in T cells. Thus, in another embodiment, antibodies that preventPD-1 ligand from providing a costimulatory signal can inhibit T cellcostimulation.

These modulatory antibodies can be administered in vitro (e.g., bycontacting the cell with an antibody) or, alternatively, in vivo (e.g.,by administering the agent to a subject). As such, the present inventionrelates to methods of treating an individual afflicted with a disease ordisorder that would benefit from modulation of an immune response, e.g.,by modulation of the interaction between a PD-1 ligand and PD-1, or a B7polypeptide.

4. Downregulation of Immune Responses

There are numerous embodiments of the invention for upregulating theinhibitory function or downregulating the costimulatory function of aPD-1 ligand to thereby downregulate immune responses. Downregulation canbe in the form of inhibiting or blocking an immune response already inprogress, or may involve preventing the induction of an immune response.The functions of activated immune cells can be inhibited bydown-regulating immune cell responses, or by inducing specific anergy inimmune cells, or both.

For example, the immune response can be downmodulated using: anti-PD-1ligand antibodies that blocks costimulation by PD-1 ligand (e.g., whilenot affecting or increasing the interaction between PD-L1 and PD-1) orwhich promote the binding of a PD-1 ligand with PD-1, (e.g., while notaffecting or while inhibiting costimulation by PD-1 ligand).

In one embodiment of the invention, tolerance is induced againstspecific antigens by co-administering an antigen with an antibody whichblocks PD-1 ligand costimulation. For example, tolerance can be inducedto specific proteins. In one embodiment, immune responses to allergens,or to foreign proteins to which an immune response is undesirable, canbe inhibited. For example, patients that receive Factor VIII frequentlygenerate antibodies against this clotting factor. Co-administration ofan antibody that blocks a PD-1 ligand-mediated costimulatory signal oran antibody that stimulates a PD-1 mediated inhibitory signal incombination with recombinant factor VIII (or by physically linked toFactor VIII, e.g., by cross-linking) can result in downmodulation.

In one embodiment, two separate agents that downmodulate immuneresponses can be combined as a single composition or administeredseparately (simultaneously or sequentially) to more effectivelydownregulate immune cell mediated immune responses in a subject.Furthermore, a therapeutically active amount of one or more of thesubject antibodies, can be used in conjunction with other downmodulatingreagents to influence immune responses. Examples of otherimmunomodulating reagents include, without limitation, antibodies thatblock a costimulatory signal, (e.g., against CD28 or ICOS), antibodiesthat act as agonists of CTLA4, and/or antibodies against other immunecell markers (e.g., against CD40, against CD40 ligand, or againstcytokines), fusion proteins (e.g., CTLA4-Fc), and immunosuppressivedrugs, (e.g., rapamycin, cyclosporine A or FK506).

Downregulating or preventing a PD-1 ligand costimulation, or promotingan interaction between a PD-1 ligand and PD-1 is useful to downmodulatethe immune response, e.g., in situations of tissue, skin and organtransplantation, in graft-versus-host disease (GVHD), or in inflammatorydiseases such as systemic lupus erythematosus, and multiple sclerosis.For example, blockage of immune cell function results in reduced tissuedestruction in tissue transplantation. Typically, in tissue transplants,rejection of the transplant is initiated through its recognition asforeign by immune cells, followed by an immune reaction that destroysthe transplant. The administration of an antibody which inhibits PD-1ligand costimulation alone or in conjunction with another downmodulatoryagent, prior to or at the time of transplantation can promote thegeneration of an inhibitory signal. Moreover, inhibition of PD-1 ligandcostimulatory signals, or promotion of a PD-1 ligand or PD-1 inhibitorysignals, may also be sufficient to anergize the immune cells, therebyinducing tolerance in a subject. Induction of long-term tolerance byblocking a PD-1 ligand mediated costimulatory signal may avoid thenecessity of repeated administration of these blocking reagents.

To achieve sufficient immunosuppression or tolerance in a subject, itmay also be desirable to block the costimulatory function of otherpolypeptides. For example, it may be desirable to block the function ofB7-1, B7-2, or B7-1 and B7-2 by administering a soluble form of acombination of peptides having an activity of each of these antigens,blocking antibodies against these antigens or blocking small molecules(separately or together in a single composition) prior to or at the timeof transplantation. Alternatively, it may be desirable to promoteinhibitory activity of a PD-1 ligand or PD-1 and inhibit a costimulatoryactivity of B7-1 and/or B7-2. Other downmodulatory agents that can beused in connection with the downmodulatory methods of the inventioninclude, for example, agents that transmit an inhibitory signal viaCTLA4, soluble forms of CTLA4, antibodies that activate an inhibitorysignal via CTLA4, blocking antibodies against other immune cell markersor soluble forms of other receptor ligand pairs (e.g., agents thatdisrupt the interaction between CD40 and CD40 ligand (e.g., anti CD40ligand antibodies)), antibodies against cytokines, or immunosuppressivedrugs.

Downmodulation of immune responses are also useful in treatingautoimmune disease. Many autoimmune disorders are the result ofinappropriate activation of immune cells that are reactive against selftissue and which promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive immune cells may reduce or eliminate disease symptoms.Administration of reagents which block costimulation of immune cells bydisrupting interactions between PD-1 ligand and B7 polypeptides, or bypromoting the interaction between PD-1 ligand and PD-1, withoutmodulating or while downmodulating the interaction between PD-1 ligandand a B7 polypeptide, are useful for inhibiting immune cell activationand preventing production of autoantibodies or cytokines which may beinvolved in the disease process. Additionally, agents that promote aninhibitory function of a PD-1 ligand or PD-1 may induce antigen-specifictolerance of autoreactive immune cells, which could lead to long-termrelief from the disease. The efficacy of reagents in preventing oralleviating autoimmune disorders can be determined using a number ofwell-characterized animal models of human autoimmune diseases. Examplesinclude murine experimental autoimmune encephalitis, systemic lupuserythematosus in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmunecollagen arthritis, diabetes mellitus in NOD mice and BB rats, andmurine experimental myasthenia gravis (see, e.g., Paul ed., FundamentalImmunology, Raven Press, New York, Third Edition 1993, chapter 30).

Inhibition of immune cell activation is useful therapeutically in thetreatment of allergy and allergic reactions, e.g., by inhibiting IgEproduction. An antibody that promotes a PD-1 ligand or PD-1 inhibitoryfunction can be administered to an allergic subject to inhibit immunecell mediated allergic responses in the subject Inhibition of PD-1ligand costimulation of immune cells or stimulation of a PD-1 ligand orPD-1 inhibitory pathway can be accompanied by exposure to allergen inconjunction with appropriate MHC polypeptides. Allergic reactions can besystemic or local in nature, depending on the route of entry of theallergen and the pattern of deposition of IgE on mast cells orbasophils. Thus, inhibition of immune cell mediated allergic responseslocally or systemically by administration of an inhibitory form of anagent that inhibits the interaction of a PD-1 ligand with acostimulatory receptor, or an antibody that promotes an inhibitoryfunction of a PD-1 ligand or PD-1.

Inhibition of immune cell activation through blockage of PD-1 ligandcostimulation, or through promotion of the interaction between a PD-1ligand and PD-1, may also be important therapeutically in viralinfections of immune cells. For example, in the acquired immunedeficiency syndrome (AIDS), viral replication is stimulated by immunecell activation. Modulation of these interactions may result ininhibition of viral replication and thereby ameliorate the course ofAIDS. Modulation of these interactions may also be useful in promotingthe maintenance of pregnancy. PD-1 ligand is normally highly expressedin placental trophoblasts, the layer of cells that forms the interfacebetween mother and fetus and may play a role in preventing maternalrejection of the fetus. Females at risk for spontaneous abortion (e.g.,those who have previously had a spontaneous abortion or those who havehad difficulty conceiving) because of immunologic rejection of theembryo or fetus can be treated with agents that modulate theseinteractions.

Downregulation of an immune response by modulation of PD-1 ligandcostimulation or by modulation of PD-1 ligand/PD-1 binding may also beuseful in treating an autoimmune attack of autologous tissues. Forexample, PD-1 ligand is normally highly expressed in the heart and mayprotect the heart from autoimmune attack. This is evidenced by the factthat the Balb/c PD-1 knockout mouse exhibits massive autoimmune attackon the heart with thrombosis. Thus, conditions that are caused orexacerbated by autoimmune attack (e.g., in this example, heart disease,myocardial infarction or atherosclerosis) may be ameliorated or improvedby modulation of these interactions. It is therefore within the scope ofthe invention to modulate conditions exacerbated by autoimmune attack,such as autoimmune disorders (as well as conditions such as heartdisease, myocardial infarction, and atherosclerosis).

5. Upregulation of Immune Responses

Also useful therapeutically is the blockage of the interaction of a PD-1ligand with PD-1 or B7-1 as a means of upregulating an immune response.Upregulation of immune responses can be in the form of enhancing anexisting immune response or eliciting an initial immune response. Forinstance, enhancing an immune response using the subject compositionsand methods is useful in cases of infections with microbes (e.g.,bacteria, viruses, or parasites). In one embodiment, an antibody thatblocks the interaction of a PD-1 ligand with PD-1 is used to enhance theimmune response. Such an antibody (e.g., a non-activating antibody thatblocks PD-L1 binding to PD-1) is therapeutically useful in situationswhere upregulation of antibody and cell-mediated responses would bebeneficial. Exemplary disorders include viral skin diseases, such asHerpes or shingles, in which case such an agent can be deliveredtopically to the skin. In addition, systemic viral diseases such asinfluenza, the common cold, and encephalitis might be alleviated bysystemic administration of such agents.

Alternatively, immune responses can be enhanced in an infected patientthrough an ex vivo approach, for instance, by removing immune cells fromthe patient, contacting immune cells in vitro with an antibody thatblocks the interaction of a PD-1 ligand with PD-1 and reintroducing thein vitro stimulated immune cells into the patient.

In certain instances, it may be desirable to further administer otheragents that upregulate immune responses, for example, forms of other B7family members that transduce signals via costimulatory receptors, inorder to further augment the immune response.

An antibody that blocks the interaction of a PD-1 ligand with PD-1 orB7-1 can be used prophylactically in vaccines against variouspolypeptides (e.g., polypeptides derived from pathogens). Immunityagainst a pathogen (e.g., a virus) can be induced by vaccinating with aviral protein along with an antibody that blocks the interaction of aPD-1 ligand with PD-1 or B7-1 in an appropriate adjuvant.

In another embodiment, upregulation or enhancement of an immune responsefunction, as described herein, is useful in the induction of tumorimmunity

In another embodiment, the immune response can be stimulated by themethods described herein, such that preexisting tolerance is overcome.For example, immune responses against antigens to which a subject cannotmount a significant immune response, e.g., to an autologous antigen,such as a tumor specific antigens can be induced by administering anantibody that blocks the interaction of a PD-1 ligand with PD-1. In oneembodiment, an autologous antigen, such as a tumor-specific antigen canbe coadministered. In another embodiment, an immune response can bestimulated against an antigen (e.g., an autologous antigen) to treat aneurological disorder. In another embodiment, the subject agents can beused as adjuvants to boost responses to foreign antigens in the processof active immunization.

In one embodiment, immune cells are obtained from a subject and culturedex vivo in the presence of an antibody as described herein, to expandthe population of immune cells and/or to enhance immune cell activation.In a further embodiment the immune cells are then administered to asubject Immune cells can be stimulated in vitro by, for example,providing to the immune cells a primary activation signal and acostimulatory signal, as is known in the art. Various agents can also beused to costimulate proliferation of immune cells. In one embodimentimmune cells are cultured ex vivo according to the method described inPCT Application No. WO 94/29436. The costimulatory polypeptide can besoluble, attached to a cell membrane, or attached to a solid surface,such as a bead.

Other embodiments of the present invention are described in thefollowing Examples. The present invention is further illustrated by thefollowing examples which should not be construed as further limiting.The contents of Sequence Listing, figures and all references, patentsand published patent applications cited throughout this application areexpressly incorporated herein by reference.

EXAMPLES

The examples below describe the generation of monoclonal antibodiessuitable for therapeutic purposes targeting human PD-1, PD-L1 and PD-L2.Composite, human anti-human PD-1, PD-L1 and PD-L2 antibodies weregenerated from mouse anti-human EH12.2H7, 29E.2A3 and 24F.10C12antibodies, respectively. Segments of human V region sequence weresourced from unrelated human antibody (germline and non-germline)sequence databases. Each selected sequence segment (as well as thejunctions between segments) was tested for the potential to bind to MHCclass II using binding prediction algorithms. All final composite, humanantibody sequence variants were designed to avoid T cell epitopes.Composite, human antibody V region genes were generated using syntheticoligonucleotides encoding combinations of the human sequence segments.These were then cloned into vectors containing human constant regions,and antibodies were produced and tested for binding to target antigensby competition ELISA.

Example 1 Design of Composite, Human Antibody Variable Region Sequences

Structural models of the mouse EH12.2H7, 29E.2A3 and 24F.10C12 V regionswere produced using Swiss Pdb and analyzed in order to identifyimportant “constraining” amino acids in the mouse V regions that mightbe essential for the binding properties of the antibodies. Only residuescontained within the CDRs were considered to be important, including CDRresidues defined under both Kabat and Chothia definitions.

From the above analysis, it was considered that composite, human formsof EH12.2H7, 29E.2A3 and 24F.10C12 could be created with wide latitudeof sequences outside of CDRs but with a narrow menu of possiblealternative residues within the CDR sequences. Preliminary analysisindicated that corresponding sequence segments from several humanantibodies could be combined to create CDRs similar or identical tothose in the mouse sequences. For regions outside of and flanking theCDRs, a wide selection of human sequence segments were identified aspossible components of the novel composite, human antibody variableregions.

Based upon the above analysis, a large preliminary set of sequencesegments that could be used to create EH12.2H7, 29E.2A3 and 24F.10C12composite, human antibody variants were selected and analyzed via MHCclass II binding prediction algorithms and BLAST searched through aproprietary database of known antibody sequence related T cell epitopes.Sequence segments where potential MHC class II binding peptides wereidentified, or scored significant hits against the database of known Tcell epitopes, were discarded. This resulted in a reduced set ofsegments, and combinations of these were again analyzed, as above, toensure that junctions between segments did not contain potential T cellepitopes. Selected segments were then combined to produce heavy andlight chain variable region sequences for synthesis. For all threeantibodies, five heavy chains and four light chains were constructedwith sequences detailed as follows;

Antigen Composite VH Sequences Composite VK Sequences PD-1 FIG. 2 (A-E)FIG. 3 (A-D) PD-L1 FIG. 4 (A-E) FIG. 5 (A-D) PD-L2 FIG. 6 (A-E) FIG. 7(A-D)

Sequence segments used to produce these composite, human antibodysequences are detailed in Tables 1, 2, and 3 for antibodies againstPD-1, PD-L1 and PD-L2 respectively.

TABLE 1 Derivation of Human Sequence Segments thatComprise the Anti-PD-1 Composite, Human Antibodies (a) Genbank VH3Accession No. Sequence BAA75018 QVQLVQSGHEVKQPGASVK (SEQ ID NO: 82)AAG00910 MSCKASGYSFTS (SEQ ID NO: 83) AAY18543SGYSFTSSWI (SEQ ID NO: 84) AAY57105 WIHWV (SEQ ID NO: 85) AAG00910 KQAAD16517 QAPGQGLEWIG (SEQ ID NO: 86) AAD53797GLEWIGYIYPS (SEQ ID NO: 87) CAA08742 STGF (SEQ ID NO: 88) CAC87219TEYN (SEQ ID NO: 89) AAT96419 QKF AAA17939 KDR AAR02530DRAT (SEQ ID NO: 90) AAA17939 TLT AAM87977 TADKSTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 91) CAA78534 STAYMELSSLRSEDTAVYYCARWRD  (SEQ ID NO: 92)AAV40096 DSSGY (SEQ ID NO: 93) AAR38557 YHA AAW29142 AMD IGHJ4DYWGQGTLVTVSS (SEQ ID NO: 94) (b) Genbank VH4 Accession No. SequenceBAA75018 QVQLVQSGHEVKQPGASVK (SEQ ID NO: 82) AAG00910MSCKASGYSFTS (SEQ ID NO: 83) AAY18543 SGYSFTSSWI (SEQ ID NO: 84)AAA02616 HWVRQAPGQGLEWIG (SEQ ID NO: 95) AAD53797GLEWIGYIYPS (SEQ ID NO: 87) CAA08742 STGF (SEQ ID NO: 88) CAC87219TEYN (SEQ ID NO: 89) AAT96419 QKF AAA17939 KDR AAR02530DRAT (SEQ ID NO: 90) AAA17939 TLT AAM87977 TADKSTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 91) CAA78534 STAYMELSSLRSEDTAVYYCARWRD  (SEQ ID NO: 92)AAV40096 DSSGY (SEQ ID NO: 93) AAR38557 YHA AAW29142 AMD IGHJ4DYWGQGTLVTVSS (SEQ ID NO: 94) (c) Genbank Vκ3 Accession No. SequenceAAY16615 EIVLTQSPATLSLSPGQR (SEQ ID NO: 96) AAD09377RLTISCRASQ (SEQ ID NO: 97) AAA99362 TISCRASQSVST (SEQ ID NO: 98)AAL04518 SVSTSGYSYMHW (SEQ ID NO: 99) AAA58912WYQQKPDQSPKLLIK (SEQ ID NO: 100) AAD16648 FGS AAD19478SNLESG (SEQ ID NO: 101) AAL10884 GIPARFSGSGSGTDFTLTISSLEPEDFA (SEQ ID NO: 102) AAD16559 PEDFATYYCQHS (SEQ ID NO: 103) AAA99326 SWAAC16811 EIP human J2 YTFGQGTKLEIK (SEQ ID NO: 104) (d) Genbank Vκ4Accession No. Sequence AAB53267 DIVLTQSP (SEQ ID NO: 105) AAY16615IVLTQSPATLSLSPGQR (SEQ ID NO: 106) AAD09377 RLTISCRASQ (SEQ ID NO: 97)AAA99362 TISCRASQSVST (SEQ ID NO: 98) AAL04518SVSTSGYSYMHW (SEQ ID NO: 99) AAA58912 WYQQKPDQSPKLLIK (SEQ ID NO: 100)AAD16648 FGS AAD19478 SNLESG (SEQ ID NO: 101) AAL10884GIPARFSGSGSGTDFTLTISSLEPEDFA  (SEQ ID NO: 102) AAD16559PEDFATYYCQHS (SEQ ID NO: 103) AAA99326 SW AAC16811 EIP human J2YTFGQGTKLEIK (SEQ ID NO: 104)

TABLE 2 Derivation of Human Sequence Segments that Comprise the Anti-PD-L1 Composite,Human  Antibodies (a) Genbank VH2Accession No. Sequence ABI50688 EVQLVQSGAEVKKPGASVK (SEQ ID NO: 107)AAG00910 MSCKASGY (SEQ ID NO: 108) ABI50688SCKASGYTFTSY (SEQ ID NO: 109) AAC50839 SYVMHWV (SEQ ID NO: 110) CAC43594WVKQ (SEQ ID NO: 111) AAA18267 QAPGQRLEWIG (SEQ ID NO: 112) ABF20472 GYAAD30737 VNPF (SEQ ID NO: 113) CAL06274 NDGT (SEQ ID NO: 114) CAC43212KYN CAC87219 YNE CAD31770 EM AAR32413 FKGR (SEQ ID NO: 115) AAG30515GRAT (SEQ ID NO: 116) ABA62048 TLT ABI50549 TSD AAR32572DKSTSTAYMELSSLRSEDTAVYYCA  (SEQ ID NO: 117) AAC18225AVYYCARQA (SEQ ID NO: 118) AAV39747 AWGY (SEQ ID NO: 119) IGHJ5*02PWGQGTLVTVSS (SEQ ID NO: 120) (b) Genbank VH4 Accession No. SequenceABI50688 EVQLVQSGAEVKKPGASVK (SEQ ID NO: 107) AAG00910MSCKASGY (SEQ ID NO: 108) ABI50688 SCKASGYTFTSY (SEQ ID NO: 109)AAC50839 SYVMHWV (SEQ ID NO: 110) AAA18267WVRQAPGQRLEWIG (SEQ ID NO: 121) ABF20472 GY AAD30737VNPF (SEQ ID NO: 113) CAL06274 NDGT (SEQ ID NO: 114) CAC43212 KYNCAC87219 YNE CAD31770 EM AAR32413 FKGR (SEQ ID NO: 115) AAG30515GRAT (SEQ ID NO: 116) ABA62048 TLT ABI50549 TSD AAR32572DKSTSTAYMELSSLRSEDTAVYYCA  (SEQ ID NO: 117) AAC18225AVYYCARQA (SEQ ID NO: 118) AAV39747 AWGY (SEQ ID NO: 119) IGHJ5*02PWGQGTLVTVSS (SEQ ID NO: 120) (c) Genbank Vκ1 Accession No. SequenceCAA31193 DIVLTQSPASLALS (SEQ ID NO: 122) ABA26115LSPGERAT (SEQ ID NO: 123) AAQ21828 ESV CAA51101 VE AAA58691YYGTSL (SEQ ID NO: 124) AAY33369 VQWYQQKPGQ (SEQ ID NO: 125) ABI74051WYQQKPGQPPKLLIY (SEQ ID NO: 126) CAC39383 PKLLIYAASS (SEQ ID NO: 127)CAA38592 SVDS (SEQ ID NO: 128) AAK26833 DSGVPSRFSGSGSGT (SEQ ID NO: 129)AAM46660 RFSGSGSGTDFTLTINSLE (SEQ ID NO: 130) AAL04518EEEDAA (SEQ ID NO: 131) AAK68016 AMYFCQQ (SEQ ID NO: 132) CAK50767 SRAAP23227 RVPYTFG (SEQ ID NO: 133) Human J2 YTFGQGTKLEIK (SEQ ID NO: 104)(d) Genbank Vκ2 Accession No. Sequence CAA31193DIVLTQSPASLALS (SEQ ID NO: 122) CAE54363IVLTQSPATLSLSPGE (SEQ ID NO: 134) ABA26115 LSPGERAT (SEQ ID NO: 123)AAQ21828 ESV CAA51101 VE AAA58691 YYGTSL (SEQ ID NO: 124) AAY33369VQWYQQKPGQ (SEQ ID NO: 125) ABI74051 WYQQKPGQPPKLLIY (SEQ ID NO: 126)CAC39383 PKLLIYAASS (SEQ ID NO: 127) CAA38592 SVDS (SEQ ID NO: 128)AAK26833 DSGVPSRFSGSGSGT (SEQ ID NO: 129) AAM46660RFSGSGSGTDFTLTINSLE (SEQ ID NO: 130) AAA58912TINSLEAEDAA (SEQ ID NO: 135) AAK68016 AMYFCQQ (SEQ ID NO: 132) CAK50767SR AAP23227 RVPYTFG (SEQ ID NO: 133) Human J2YTFGQGTKLEIK (SEQ ID NO: 104) (e) Genbank Vκ4 Accession No. SequenceCAA31193 DIVLTQSPASLALS (SEQ ID NO: 122) CAE54363IVLTQSPATLSLSPGE (SEQ ID NO: 134) ABA26115 LSPGERAT (SEQ ID NO: 123)AAQ21828 ESV CAA51101 VE AAA58691 YYGTSL (SEQ ID NO: 124) AAY33369VQWYQQKPGQ (SEQ ID NO: 125) ABI74051 WYQQKPGQPPKLLIY (SEQ ID NO: 126)CAC39383 PKLLIYAASS (SEQ ID NO: 127) CAA38592 SVDS (SEQ ID NO: 128)AAK26833 DSGVPSRFSGSGSGT (SEQ ID NO: 129) AAM46660RFSGSGSGTDFTLTINSLE (SEQ ID NO: 130) AAA58912TINSLEAEDAATYFC (SEQ ID NO: 136) AAK68016 AMYFCQQ (SEQ ID NO: 132)CAK50767 SR AAP23227 RVPYTFG (SEQ ID NO: 133) Human J2YTFGQGTKLEIK (SEQ ID NO: 104)

TABLE 3 Derivation of Human Sequence Segments that Comprise the Anti-PD-L2 Composite, Human  Antibodies (a) Genbank VH2Accession No. Sequence ABF83419 QVQLVQSGAEVKKPGASVK (SEQ ID NO: 137)AAG00910 MSCKASGY (SEQ ID NO: 108) ABF83419SCKASGYTFTGY (SEQ ID NO: 138) AAL17955 TMHWV (SEQ ID NO: 139) CAC43594WVKQ (SEQ ID NO: 111) AAL17955 QAPG (SEQ ID NO: 140) AAF40162GQGLEWIG (SEQ ID NO: 141) AAR02558 GYINP (SEQ ID NO: 142) AAR32283INPRSG (SEQ ID NO: 143) AAR02553 GYT CAC87219 TEYN (SEQ ID NO: 89)AAT96419 QKF AAA17939 KDR AAB06403 RTT AAA17939 TLT AAG30529TADKSTSTAYMELSSLRSEDTAVYYCAR  (SEQ ID NO: 91) ABE66740DTAVYYCARPW (SEQ ID NO: 144) ABK81281 WFAYWGQGT (SEQ ID NO: 145) IGHJ4YWGQGTLVTVSS (SEQ ID NO: 146) (b) Genbank VH4 Accession No. SequenceABF83419 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGY (SEQ ID NO: 147) AAL17955TMHWVRQAPG (SEQ ID NO: 148) AAF40162 GQGLEWIG (SEQ ID NO: 141) AAR02558GYINP (SEQ ID NO: 142) AAR32283 INPRSG (SEQ ID NO: 143) AAR02553 GYTCAC87219 TEYN (SEQ ID NO: 89) AAT96419 QKF AAA17939 KDR AAB06403 RTTAAA17939 TLT AAG30529 TADKSTSTAYMELSSLRSEDTAVYYCAR  (SEQ ID NO: 91)ABE66740 DTAVYYCARPW (SEQ ID NO: 144) ABK81281WFAYWGQGT (SEQ ID NO: 145) IGHJ4 YWGQGTLVTVSS (SEQ ID NO: 146) (c)Genbank Vκ2 Accession No. Sequence AAD16249 DIVMTQSP (SEQ ID NO: 149)CAA31193 PASL (SEQ ID NO: 150) AAA58913 LSVTPGEKVTITC (SEQ ID NO: 151)AAQ99244 CKSSQSLL (SEQ ID NO: 152) ABA71421 LNS AAD19451 GN AAS86065 QKAAD14073 KNYLTWYQQKPGQPPKLLIYWASTRESGVPDRF (SEQ ID NO: 153) AAZ09126RFTGSGSGTDFTLTISSLQAEDVAVYYCQ  (SEQ ID NO: 154) CAA31484 NDY CAC87582YSYPL (SEQ ID NO: 155) human J1 TFGQGTKLEIK (SEQ ID NO: 156) (d) GenbankVκ3 Accession No. Sequence AAD16249 DIVMTQSP (SEQ ID NO: 149) AAA58913VMTQSPAFLSVTPGEKVTITC  (SEQ ID NO: 157) AAQ99244CKSSQSLL (SEQ ID NO: 152) ABA71421 LNS AAD19451 GN AAS86065 QK AAD14073KNYLTWYQQKPGQPPKLLIYWASTRESGVPDRF (SEQ ID NO: 153) AAZ09126RFTGSGSGTDFTLTISSLQAEDVAVYYCQ  (SEQ ID NO: 154) CAA31484 NDY CAC87582YSYPL (SEQ ID NO: 155) human J1 TFGQGTKLEIK (SEQ ID NO: 156) (e) GenbankVκ4 Accession No. Sequence AAD16249 DIVMTQSP (SEQ ID NO: 149) AAA58913VMTQSPAFLSVTPGEKVTITC  (SEQ ID NO: 157) AAQ99244CKSSQSLL (SEQ ID NO: 152) ABA71421 LNS AAD19451 GN AAS86065 QK AAD14073KNYLTWYQQKPGQPPKLLIYWASTRESGVPDRF (SEQ ID NO: 153) CAD44754RFSGSGSGTDFTLTISSLQAEDVAVYYCQ  (SEQ ID NO: 158) CAA31484 NDY CAC87582YSYPL (SEQ ID NO: 155) human J1 TFGQGTKLEIK (SEQ ID NO: 156)

Example 2 Generation and Testing of Composite, Human Antibodies

Initial variant 1 composite, human antibody VH and VK region genes weresynthesized for EH12.2H7, 29E.2A3 and 24F.10C12 using a series ofoverlapping oligonucleotides that were annealed, ligated and PCRamplified to give full length synthetic V-regions (FIG. 2A, FIG. 3A,FIG. 4A, FIG. 5A, FIG. 6A and FIG. 7A). For each composite, humanantibody, subsequent sequence variants were constructed using longoverlapping oligonucleotides and PCR, using the initial variant 1 as thetemplate. The assembled variants were then cloned directly intoexpression vectors (FIG. 1) and their sequences were verified.

All combinations of chimeric and composite heavy and light chains (i.e.a total of 20 pairings for each antibody) were stably transfected intoNS0 cells by electroporation and selected in media (high glucose DMEMwith L-glutamine and Na pyruvate, 5% ultra-low IgG FCS, pen/strep-allfrom Invitrogen) containing 200 nM methotrexate. Several drug resistantcolonies for each construct were tested for expression levels and thebest expressing lines were selected and frozen under liquid nitrogen.

Supernatants from the best expressing lines for each combination werequantified using an Fc capture, Kappa light chain detection ELISA incomparison to a IgG1/kappa standard. The quantified supernatants werethen tested in a competition ELISA for binding to their target antigen.Ninety-six well Maxisorb™ plates (Nunc) were coated overnight at 4° C.with 50 μl/well of 1 μg/ml human Fc-PD-1, Fc-PD-L1 or Fc-PD-L2 (R&Dsystems) in carbonate buffer pH 9.6. Duplicate titrations of mousereference antibody and composite, human antibody samples were generated(in the range 0.0078 μg/ml to 8 μg/ml) and mixed with a constantconcentration (40 ng/ml) of biotinylated mouse reference antibody in PBSpH 7.4/2% BSA. The titrations, 100 μl/well, were added to washed (4×with PBS pH 7.4/0.05% Tween 20) assay plates and incubated at roomtemperature for 1 hour. Plates were washed as above and 100 μl/well of a1/1000 dilution of streptavidin HRP (Sigma) in PBS pH 7.4/2% BSA wasadded and incubated for a further 1 hour at room temperature. After afurther wash, bound biotinylated reference antibody was detected with100 μl/well OPD substrate. Absorbance was measured at 490 nm and thebinding curves of the test antibodies were compared to the mousereference standard. Absorbance was plotted against sample concentrationand straight lines were fitted through each of the data sets. Theequations of the lines were used to calculate the concentration requiredto inhibit Biotin-EH12.2H7 binding to PD-1, Biotin-29E.2A3 binding toPD-L1 and Biotin-24F.10C12 binding to human PD-L2 by 50% (IC₅₀).

The antibodies with the best IC₅₀ were selected and cell lines for allthese variants of EH12.2H7, 29E.2A3 and 24F.10C12 antibodies were bulkedup to 100 ml and grown to saturation. Antibodies were purified from eachculture via protein A affinity chromatography. Briefly, supernatantswere pH adjusted with 0.1 volume of 10×PBS pH 7.4 and passed over 1 mlMab Select Sure protein A columns (GE Healthcare). The columns werewashed with 10 volumes of PBS pH 7.4 before elution with 50 mM citratebuffer pH 3.0. 1 ml fractions were collected and immediately neutralizedwith 0.1 ml of 1 m Tris-HCl pH 9.0. Protein containing fractions (asjudged by absorbance at 280 nm) were pooled, buffer exchanged into PBSpH 7.4 and the purified antibodies stored at +4° C. FIGS. 8A-C shows aSDS-PAGE gel of 1 μg of each antibody, stained with coomassie blue. Theconcentrations of the antibodies were calculated by UV absorption basedupon calculated molar extinction coefficients such that E_(0.1%) at 280nm=1.61 for EH12.2H7, E_(0.1%) at 280 nm=1.46 for 29E.2A3 and E_(0.1%)at 280 nm=1.57 for 24F.10C12.

The purified antibodies were tested for binding to human Fc-PD-1,Fc-PD-L1 or Fc-PD-L2 via competition ELISA as described above.Titrations of the test antibodies were done from 0.0625 μg/ml to 8.0μg/ml in duplicate. Absorbance at 490 nm was measured and this wasplotted against test antibody concentration (FIGS. 9A-C, 10A-C).

Table 4 summarizes the results for the combinations of the composite VHand VK variant sequences for the anti-PD-1, PD-L1 and PD-L2 antibodies.For EH12.2H7 all the humanized antibodies have an IC50 that is improvedcompared to the mouse reference, particularly VH4/VK3 that has atwo-fold increase in binding. In the case of 29E.2A3, variants VH2/VK1and VH2/VK4 have equivalent binding to the mouse reference whereasvariants VH2/VK2 and VH4/VK2 have reduced binding by 1.75 and 1.36 foldrespectively. For 24F.10C12, all selected variants have similar, butslightly reduced, binding compared to the mouse reference (1.13 fold).

TABLE 4 IC50 Values for PD-1, PD-L1 and PD-L2 Composite, human AntibodySequence Variants EH12.2H7 29E.2A3 24F.10C12 IC50 IC50 IC50 Antibodyμg/ml Antibody μg/ml Antibody μg/ml mouse 1.23 mouse 0.28 mouse 0.52VH3/VK3 0.93 VH2/VK1 0.27 VH2/VK2 0.58 VH3/VK4 0.74 VH2/VK2 0.49 VH2/VK30.59 VH4/VK3 0.57 VH4/VK2 0.38 VH4/VK2 0.60 VH4/VK4 0.91 VH2/VK4 0.29VH4/VK4 0.58

As a result of these experiments, composite, human antibodies specificfor human PD-1, PD-L1 and PD-L2 have been constructed from amino acidsequence segments derived entirely from unrelated human antibodyvariable regions. All CDR and framework regions in the composite, humanantibody variants comprised more than one unrelated human sequencesegment (sourced from the human sequence database), and all composite,human antibodies were designed specifically to avoid T cell epitopes.Four lead candidates were initially selected for binding to human PD-1,PD-L1 or PD-L2 and, upon subsequent analysis, were demonstrated to havebinding within two-fold of the murine antibody.

Example 5 Enhanced Stimulation of T Cell Activation by Inhibition ofPD-1:PD-Ligand Interaction

The PD-1 signaling pathway inhibits moderate TCR/CD28 costimulatorysignals, with cytokine production being reduced first without a decreasein T cell proliferation. As the TCR/CD28 costimulatory signals weaken,the PD-1 pathway dominates, with a great reduction in cytokineproduction accompanied by a reduction in proliferation. Accordingly, inorder to confirm that the inhibition of the PD-1 pathway via inhibitionof the interaction with PD-L1 or PD-L2 using composite, human antibodiesof the invention enhances T cell activation, mixed lymphocyte reactions(MLRs) are performed.

Immature myeloid dendritic cells are isolated by culturing humanperipheral blood monocytes in IL-4 and GM-CSF. Exposure of immaturedendritic cells to an inflammatory cocktail of IL-1β, TNF-α, IL-6, andPGE₂ elicits the development of mature dendritic cells that function asAPCs. However, the addition of IL-10 to the inflammatory cytokines givenduring the maturation phase results in APCs that function only ⅙ to ⅓ aswell.

T cell activation assays (MLRs) are performed, using IL-10 treateddendritic cells as APCs, in the presence of composite, human antibodiesto PD-1, PD-L1 and/or PD-L2, or control antibodies. The addition ofanti-PD-1, anti-PD-L1 and/or PD-L2 mAb to cultures of IL-1 0 treateddendritic cells plus allogeneic T cells is predicted to result in anincrease in T cell proliferation and cytokine expression, as compared tocontrol IgG treated cultures. A combination of anti-PD-1 antibodies withanti-PD-L1 antibodies, anti-PD-L2 antibodies, may also result in anincrease in stimulation greater than that seen with either antibodyalone.

Example 6 Inhibition of the PD-1 Pathway in Chronically-Infected Mice

Mice infected with various strains of the lymphocytic choriomeningitisvirus (LCMV) are used to study the effect of chronic viral infection onCD8 T cell function. The LCMV Armstrong strain causes an acute infectionthat is cleared within 8 days, leaving behind a long-lived population ofhighly functional, resting memory CD8 T cells. The LCMV Cl-13 strain, incontrast, establishes a persistent infection in the host, characterizedby a viremia that lasts up to 3 months.

To confirm that blocking the PD-1 signaling restores T cell function andenhances viral control during chronic LCMV infection, the PD-1signalling is disrupted during chronic LCMV infection using composite,human anti-PD-1 antibodies, anti-PD-L1 antibodies and/or anti-PD-L2antibodies of the invention. The antibodies are administered every thirdday to mice infected with LCMV Cl-13 from day 23 to day 37post-infection. It is expected that at day 37 there will be several-foldmore LCMV specific CD8 T cells in treated mice relative to the untreatedcontrols. It is also expected that the induction of proliferation willbe specific to CD8 T cells since and the number of CD4 T cells in thespleen will probably be approximately the same in both treated mice anduntreated mice.

In addition to an increase in CD8 T cell proliferation, it is expectedthat the inhibition of PD-1 signaling will also result in an increasedproduction of anti-viral cytokines in virus-specific CD8 T cells. Theproduction of IFN-gamma and TNF-alpha by CD8 T cells will likely beseveral-fold higher in treated mice as compared to untreated mice. Viralclearance should also be accelerated, and reduced viral titers should beobserved in the lung and kidney by day 37 post-infection in treatedmice, while untreated mice likely will display significant levels ofvirus in all these tissues.

CD4 T cells play a key role in the generation and maintenance of CD8 Tcell responses. In this regard, CD8 T cells primed in the absence of CD4T cells are incapable of mounting normal immune responses, and are thusoften referred to as “helpless T cells.” Furthermore, chronic LCMVinfection is more severe in the absence of CD4 T cells. Accordingly,helpless T cells generated during LCMV-Cl-13 infection display an evenmore profound functional impairment than T cells generated in thepresence of CD4 T cells.

CD4 T cells are depleted at the time of LCMV-Cl-13 infection and miceare treated with composite, human anti-PD-1 antibodies, anti-PD-L1antibodies and/or anti-PD-L1 antibodies of the present invention fromday 46 to day 60 post-infection. It is expected that followingtreatment, treated mice likely will have several-fold more LCMV-specificCD8T cells in their spleen than untreated control mice. This increase invirus-specific CD8 T cells in treated mice likely will be the result toan increase in proliferation, as detected by BrdU incorporation. BrdUanalysis is performed by introducing 1 mg/ml BrdU in the drinking waterduring treatment and staining is performed according to themanufacturer's protocol (BD Biosciences, San Diego, Calif.).

To confirm that the inhibition of PD-1 signals increases the lyticactivity of helpless, exhausted, virus-specific CD8 T cells, ex vivolytic activity of virus-specific CD8 T cells is detected followingtreatment using a ⁵¹Cr release assay (Wherry et al., 2003. J. Virol.77:4911-27). Viral titers are expected to be reduced by several-fold inthe spleen, liver, lung, and serum after 2 weeks of treatment relativeto untreated mice.

Example 7 Administration of a Vaccine with an Inhibitor of PD-1Signaling

One approach for boosting T cell responses during a persistent infectionis therapeutic vaccination. The rationale for this approach is thatendogenous antigens may not be presented in an optimal or immunogenicmanner during chronic viral infection and that providing antigen in theform of a vaccine may provide a more effective stimulus forvirus-specific T and B cells. Using the chronic LCMV model, mice areadministered a recombinant vaccinia virus expressing the LCMV GP33epitope as a therapeutic vaccine (VVGP33), which results in a modestenhancement of CD8 T cell responses in some chronically infected mice.This therapeutic vaccination is combined with composite, human anti-PD-1antibodies, anti-PD-L1 antibodies and/or anti-PD-L2 antibodies of theinvention. It is expected that LCMV specific T cell responses will beboosted to a greater level than compared to either treatment alone andthe effect of combined treatment will likely be more than additive.

Example 8 Chimpanzees as a Model for Immunotherapy of Persistent HCVInfection

Chimpanzees provide a model of HCV persistence in humans. Defects in Tcell immunity leading to life-long virus persistence both include adeficit in HCV-specific CD4 helper T cells and impaired or altered CD8effector T cell activity. Persistently infected chimpanzees are treatedwith composite, human anti-PD-1 antibodies, anti-PD-L1 antibodies and/oranti-PD-L2 antibodies of the invention. The efficacy of blockade of theinhibitory pathways, combined with vaccination using recombinantstructural and non-structural HCV proteins, and whether such strategiescan enhance the frequency and longevity of virus-specific memory T cellsare determined. The defect in T cell immunity is exclusivelyHCV-specific in persistently infected humans and chimpanzees. Antiviralactivity may then be restored by delivering to chimpanzees humanizedmonoclonal antibodies that block signaling through these molecules.

Persistently infected chimpanzees are treated with composite, humananti-PD-1 antibodies, anti-PD-L1 antibodies and/or anti-PD-L2 antibodiesof the invention. After treatment with antibodies, the humoral andcellular immune responses as well as the HCV RNA load are determined.Samples are collected at weeks 1, 2, 3, 5, and 8, and then at monthlyintervals. Samples include: 1) serum for analysis of transaminases,autoantibodies, neutralizing antibodies to HCV, and cytokine responses,2) plasma for viral load and genome evolution, 3) PBMC for in vitromeasures of immunity, costimulatory/inhibitory receptor expression andfunction, 4) fresh (unfixed) liver for isolation of intrahepaticlymphocytes and RNA, and 5) fixed (formalin/paraffin embedded) liver forhistology and immunohistochemical analysis. Regional lymph nodes arealso collected at 2 or 3 time points to assess expression ofco-inhibitory molecules and splice variants by immunohistochemistry andmolecular techniques.

To determine if vaccination with HCV antigens potentiates thetherapeutic effect of the antibodies, chimpazees are treated asfollows: 1) intramuscular immunization with recombinant envelopeglycoproteins E1 and E2 (in MF59 adjuvant) and other proteins (core plusNS 3, 4, and 5 formulated with ISCOMS) at weeks 0, 4, and 24; 2)intramuscular immunization with the vaccine used in, but co-administeredwith composite, human anti-PD-1 antibodies, anti-PD-L1 antibodies and/oranti-PD-L2 antibodies of the invention antibodies. HCV-specific T and Bcell responses are monitored at monthly intervals after immunization fora period of 1 year.

Markers examined on HCV-tetramer positive and total T cells in thisanalysis include markers of differentiation (e.g. CD45RA/RO, CD62L,CCR7, and CD27), activation (e.g. CD25, CD69, CD38, and HLA-DR),survival/proliferation (e.g. bcl-2 and Ki67), cytotoxic potential (e.g.granzymes and perforin), and cytokine receptors (CD122 and CD127). Aninteresting correlation exists between pre-therapy levels of thechemokine IP-10 and response to PEG IFN-.gamma./ribavirin. IP-10 levelsare measured to investigate a potential correlation between negativeregulatory pathways or HCV-specific T cell responses and IP-10 levels.Expression of inhibitory receptors and ligands on PBMC are performed byflow cytometry.

Example 9 Enhancing SIV-Specific Immunity In Vivo by PD-1 Blockade

Immune restoration potential of blockade of PD-1 during chronic simianimmunodeficiency virus (SIV) infection was tested in macaques. FourteenIndian rhesus macaques (Macaca mulatta) infected with SIV were studied.Eight macaques were used for the early chronic phase and were infectedintravenously with 200 50% tissue culture infectious dose (TCID₅₀) ofSIV251. Six macaques were used for the late chronic phase, three wereinfected with SIV251 intrarectally and three were infected with SIV239intravenously. All macaques, except RDb11, were negative for Mamu B08and Mamu B17 alleles. RDb11 was positive for Mamu B17 allele.

In vivo antibody treatment: Macaques were infused with either partiallyhumanized mouse anti-human PD-1 antibody (clone EH12-1540) (Dorfman etal., Am. J. Surg. Pathol. 30:802-810, 2006) or a control antibody(SYNAGIS). The anti-PD-1 antibody has mouse variable heavy chain domainlinked to human IgG1 (mutated to reduce FcR and complement binding) andmouse variable light chain domain linked to human κ. The clone EH12binds to macaque PD-1 and blocks interactions between PD-1 and itsligands in vitro. SYNAGIS is a humanized mouse monoclonal antibody(IgG1κ) specific to F protein of respiratory syncytial virus. Antibodieswere administered intravenously at 3 mg kg⁻¹ of body weight on days 0,3, 7 and 10.

Immune responses: Peripheral blood mononuclear cells from blood andlymphocytes from rectal pinch biopsies were isolated as describedpreviously (Velu et al., J. Virol. 81:5819-5828, 2007). Tetramerstaining, intracellular cytokine production, and measurements ofanti-SIV Env binding antibody were performed as described previously(Amara et al, Science 292:69-74, 2001; Kannanganat et al., J. Virol.81:8468-8476, 2007; Lai et al., Virology 369:153-167, 2007).

PD-1 blockade was performed during the early (10 weeks) as well as late(about 90 weeks) phases of chronic SIV infection. Nine macaques (fiveduring the early phase and four during the late phase) received theanti-PD-1 antibody and five macaques (three during the early phase andtwo during the late phase) received an isotype control antibody(Synagis, anti-respiratory syncytial virus (RSV)-specific).

PD-1 blockade during chronic SIV infection resulted in a rapid expansionof SIV-specific CD8 T cells in the blood of all macaques. The CD8 T-cellresponses to two immunodominant epitopes, Gag CM9 (Allen et al., J.Immunol. 160:6062-6071, 1998) and Tat SL8/TL8 (Allen et al., Nature407:386-390, 2000), were studied using major histocompatibility complex(MHC) I tetrameric complexes in seven of the anti-PD-1-antibody-treatedand three of the control-antibody-treated macaques that expressed theMamu A*01 histocompatibility molecule. Most (>98%) of the Gag-CM9tetramer-specific CD8 T cells expressed PD-1 before blockade. After PD-1blockade, the Gag-CM9 tetramer-specific CD8 T cells expanded rapidly andpeaked by 7-21 days. At the peak response, these levels were about 2.5to 11-fold higher than their respective levels on day 0 (P=0.007) andremained elevated until 28-45 days. Similar results were observed withblockade during the early as well as late phases of chronic SIVinfection. A 3-4-fold increase in the frequency of Gag-specificinterferon (IFN)-y-positive CD8 T cells was also observed by day 14after blockade in the two Mamu A*01-negative animals (RTd11 and RDb11),demonstrating that PD-1 blockade can enhance the frequency ofvirus-specific CD8 T cells that are restricted by non-Mamu A*01 alleles.Expansion of SIV-specific CD8 T cells was not observed in thecontrol-antibody treated macaques.

PD-1 blockade was also associated with a significant increase in thefrequency of virus-specific CD8 T cells that were undergoing active celldivision in vivo with improved functional quality. Consistent with therapid expansion of SIV-specific CD8 T cells, the frequency of Gag-CM9tetramer-specific CD8 cells that co-expressed Ki67 (marker forproliferating cells) also increased as early as by day 7 after blockade(P=0.01). Similarly, we observed an increase in the frequencies ofGag-CM9 tetramer-specific CD8 T cells co-expressing perforin andgranzyme B (cytolytic potential; P=0.001 and P=0.03, respectively), CD28(co-stimulation potential; P=0.001), CD127 (proliferative potential;P=0.0003) and CCR7 (lymph-node homing potential; 0.001). A transient 1.5to 2-fold increase in the frequency of tetramer-negative andKi67-positive CD8 T cells after blockade was observed. This could be dueto expansion of CD8 T cells specific to other epitopes in Gag as well asother proteins of SIV, and other chronic viral infections in theseanimals. No significant enhancement was observed for these markers inthe three control antibody-treated macaques.

No expansion was observed for Tat-TL8-specific CD8 T cells afterblockade. This could be due to viral escape from recognition byTat-TL8-specific CD8 T cells, as PD-1 blockade is known to result inexpansion of T cells only when they simultaneously receive signalsthrough T-cell receptor. To test this possibility, the viral genomespresent in the plasma just before the initiation of blockade from allthree Mamu A*01-positive macaques that were infected with SIV251 andreceived the blocking antibody during the early phase of infection weresequenced. Indeed, mutations in the viral genome corresponding to theTat TL8 epitope region were found. All these mutations either have beenshown or predicted to reduce the binding of Tat SL8/TL8 peptide to MamuA*01 MHC molecule and result in escape from recognition by theTat-SL8/TL8-specific CD8 T cells”. These results suggest that in vivoblockade of PD-1 may not result in expansion of T cells that arespecific to escape mutants of viral epitopes.

PD-1 blockade also resulted in expansion of Gag-CM9-specific CD8 T cellsat the colorectal mucosal tissue (gut), a preferential site of SIV/HIVreplication. Expansion was not observed for two of the seven macaques,although expansion was evident for one of them in blood. In contrast toblood, the expansion in gut peaked much later by day 42 and ranged from2- to 3-fold compared with their respective day 0 levels (P=0.003).Similar to blood, the Gag-CM9 tetramer-specific cells that co-expressedKi67 (P=0.01), perforin (P=0.03), granzyme B (P=0.01) and CD28 (P=0.01)also increased in the gut after blockade.

More importantly, PD-1 blockade also enhanced the functional quality ofanti-viral CD8 T cells and resulted in the generation of polyfunctionalcells capable of co-producing the cytokines IFN-y, tumour-necrosisfactor (TNF)-α and interleukin (IL)-2. On the day of initiation of PD-1blockade during the late chronic phase of infection, the frequency ofGag-specific IFN-γ-positive cells was low and they failed to co-expressTNF-α and IL-2. However, after the blockade, the frequency ofIFN-γ-positive cells increased in all four PD-1 antibody-treatedmacaques (P=0.03) and they acquired the ability to co-express TNF-α andIL-2. The expansion of IFN-γ positive cells peaked by 14-21 days and thepeak levels were 2-10-fold higher than the respective day 0 levels. Onday 21, about 16% of the total Gag-specific cells co-expressed all threecytokines, and about 30% co-expressed IFN-γ and TNF-α. This is incontrast to <1% of the total Gag-specific cells co-expressing all threecytokines (P=0.01), and about 14% co-expressing IFN-γ and TNF-α on day 0(P=0.04). Similar results were also observed after blockade during theearly chronic phase of infection.

To test the role of PD-1 in regulating B-cell function during chronicimmunodeficiency virus infections, the B-cell responses after PD-1blockade in SIV-infected macaques were characterized. Analysis of PD-1expression on different B-cell subsets before PD-1 blockade revealedpreferential expression of PD-1 by memory B cells (CD20⁺CD27⁺CD21⁻)compared to naive B cells (CD20⁺CD27⁻CD21⁺; P<0.001). In vivo blockadeof PD-1 resulted in a 2 to 8-fold increase in the titer of SIV-specificbinding antibody by day 28 after blockade (P<0.001). To understand thisfurther, experiments were carried out to the proliferation of memory Bcells in SIV-infected macaques that were treated simultaneously withanti-PD-1 antibody and anti-retroviral therapy and observed asignificant increase in Ki67+(proliferating) memory, but not naive, Bcells as early as day 3. These results demonstrate that the PD-1-PDLpathway could have a role in regulating B-cell dysfunction duringchronic SIV infection.

Neutralization assays revealed a two-fold increase in titers against theeasily neutralizable laboratory-adapted SIV251 and no increase in titersagainst hard-to-neutralize wild-type SIV251 or SIV239. In two of thenine animals treated with anti-PD-1 antibody, only a minimal (<2-fold)expansion of SIV-specific antibody after blockade. Notably, thefrequency of total memory B cells in these two animals was lower (−40%of total B cells) compared with the remaining seven animals (60-90% oftotal B cells) before blockade, indicating that the level ofSIV-specific memory B cells before blockade may determine the level ofexpansion of SIV-specific antibody after blockade.

PD-1 blockade resulted in significant reductions in plasma viraemia(P=0.03) and also prolonged the survival of SW-infected macaques(P=0.001). In two of the five macaques treated with anti-PD-1 antibodyduring the early chronic phase, viral load declined by day 10 andpersisted at or below this level until day 90. In one macaque viral loaddeclined transiently and in the remaining two macaques increasedtransiently and returned to pre-blockade levels. In contrast to theearly chronic phase, all four macaques treated with the anti-PD-1antibody during the late chronic phase showed a transient increase inviraemia by day 7, but rapidly reduced the virus load by day 21 tolevels that were below their respective day 0 levels. However, the viralRNA levels returned to pre-blockade levels by day 43. As expected, nosignificant reductions in the plasma viral loads were observed in any ofthe five macaques treated with the control antibody. By 21-28 days afterblockade, the viral RNA levels in the anti-PD-1-antibody-treated animalswere 2-10-fold lower than their respective day 0 levels (P=0.03). By day150 after the blockade, four of the five macaques in the control groupwere killed owing to AIDS-related symptoms (for example loss ofappetite, diarrhoea, weight loss), whereas all nine animals in theanti-PD-1-antibody-treated group had survived (P=0.001).

The observed initial rise in plasma viraemia levels in all of the latephase-treated and some of the early-phase-treated animals could be dueto an increase in the frequency of activated CD4 T cells. To determinethis, the percentage of Ki67-positive total CD4 T cells as well as thefrequency of SIV Gag-specific IFN-y producing CD4 T cells (preferentialtargets for virus replication”) after blockade were measured. Theseanalyses revealed a transient increase in the percentage ofKi67-positive CD4 T cells by day 7-14 after blockade (P=0.002) and thisincrease was higher in animals treated during the late phase than earlyphase of infection (P=0.015). Similarly, an increase in the frequency ofGag-specific CD4 T cells was also observed, but only in animals treatedduring the late phase of infection. No significant increases wereobserved for these activated CD4 T cells in the control-antibody-treatedmacaques. These results suggest that the activated CD4 T cells couldhave contributed to the observed initial rise in plasma viraemia levelsafter blockade.

Before initiation of PD-1 blockade, the set point viral load in plasmaand total CD4 T cells in blood and gut were similar between theanti-PD-1-antibody-treated and control-antibody treated groups. However,the frequencies of Gag CM9+ cells and Gag CM9+ cells co-expressingperforin, granzyme B or CD28 were not similar between the two treatmentgroups before in vivo blockade. This raises the possibility that thesedifferences could have contributed to the expansion of Gag CM9+ cellsafter PD-1 blockade. To study the influence of the frequency of Gag CM9+cells before blockade on their expansion after blockade, theanti-PD-1-antibody-treated group into was divided into two subgroupsbased on the frequency of Gag CM9+ cells before initiation of blockadesuch that one group has similar levels and the other group has higherlevels of Gag CM9+ cells compared with the control-antibody-treatedgroup. These subgroups were then analyzed for expansion of Gag CM9+cells after blockade. Expansion of Gag CM9+ cells was evident in bothsubgroups of animals after blockade of PD-1, irrespective of whetherthey were at low or high levels before blockade. Similar results werealso observed with subgroup analyses based on the frequency of Gag CM9+cells co-expressing molecules associated with better T-cell functionsuch as perforin, granzyme B, CCR7, CD 127 or CD28. However, a trendtowards better expansion of Gag CM9+CD28+ cells in animals with higherlevels of Gag CM9+CD28+ cells before blockade was observed, suggestingthat CD28 expression may serve as a biomarker for predicting the outcomeof in vivo PD-1 blockade.

The experiments described above demonstrate that PD-1 blockade using anantibody to PD-1 results in rapid expansion of virus-specific CD8 Tcells with improved functional quality. This enhanced T-cell immunitywas seen in the blood and also in the gut, a major reservoir of SIVinfection. PD-1 blockade also resulted in proliferation of memory Bcells and increases in SIV envelope-specific antibody. These improvedimmune responses were associated with significant reductions in plasmaviral load and also prolonged the survival of SIV-infected macaques.Blockade was effective during the early (week 10) as well as late (week90) phases of chronic infection even under conditions of severelymphopenia. These results demonstrate enhancement of both cellular andhumoral immune responses during a pathogenic immunodeficiency virusinfection by blocking a single inhibitory pathway and identify a noveltherapeutic approach for control of human immunodeficiency virusinfections.

Example 10 Enhanced Proliferation of SIV-Specific CD8 T Cells FollowingIn Vitro Blockade of the PD-1:PDL Pathway by a Humanized PD-1 Antibodyand a Humanized PD-L1 Antibody

Effect of a humanized anti-PD-1 antibody derived from EH-12.2H7 and ahumanized anti-PD-L1 antibody derived from 29E.2A3 on the proliferativecapacity of SIV Gag-specific CD8 T cells was tested in vitro. Thehumanized anti-PD-1 antibody has the heavy chain variable regionsequence of SEQ ID NO:28, and the light chain variable region sequenceof SEQ ID NO:32. The humanized anti-PD-L1 antibody has the heavy chainvariable region sequence of SEQ ID NO:35, and the light chain variableregion sequence of SEQ ID NO:42. The heavy chain constant region of thehumanized antibodies is from human IgG4 with Ser 228 to Pro mutation(from CPSCP to CPPCP) so that the antibody forms dimers, and the lightchain constant region is human kappa light chain constant region. Theamino acid numbering for Ser 228 is according to the EU numberingsystem. See Aalberse et al., Immunology 105:9-19, 2002. PBMC obtainedfrom SIV-infected macaques (between 3 months to 1.5 years afterinfection) were stained with carboxyfluorescein diacetate succinimidylester (CFSE) and stimulated either with SIV Gag peptide pool or culturemedium for 6 days in the presence or absence of a blocking antibody. Atthe end of stimulation, cells were stained for surface CD3 and CD8, andintracellular Ki-67. Cells were then acquired on a FACS Calibur andanalyzed using Flowjo software. Lymphocytes were identified based on thescatter, then CD8 T cells (CD3+, CD8+) were analyzed for co-staining forKi-67 and CFSE. Ki-67+, CFSE low cells were identified as proliferatingcells.

As shown in FIG. 14A, in vitro blockade of PD-1:PD-1 ligand pathwayusing the anti-PD-1 Ab results in a significant increase inproliferation of SIV-specific CD8 T cells responses. In vitro blockadeusing the anti-PD-L1 Ab results in a modest increase in proliferation ofSIV-specific CD8 T cells responses (FIG. 14B).

Example 11 Restoration of HCV-Specific T Cell Proliferation byIntrahepatic Mononuclear Cells from a Persistently Infected Chimpanzee

CFSE-labeled intrahepatic lymphocytes (2×10⁶) were isolated fromchimpanzee 1564 that had been chronically infected with the genotype 1aH77 strain of HCV for more than 10 years. The intrahepatic lymphocyteswere co-cultured for 6 days with 4×10⁶ irradiated autologousCD8-depleted PBMC that were either unmanipulated or pulsed withoverlapping peptides spanning the entire HCV polyprotein (genotype 1aH77 strain). Cells were cultured in RPMI media supplemented withL-glutamine and 10% FCS, with and without an anti-PD-L1 blockingantibody (10 μg/ml, added at day 0 and day 2). The humanized anti-PD-L1antibody has the heavy chain variable region sequence of SEQ ID NO:35,and the light chain variable region sequence of SEQ ID NO:42. The heavychain constant region of the humanized antibodies is from human IgG4with Ser 228 to Pro mutation (from CPSCP to CPPCP) so that the antibodyforms dimers, and the light chain constant region is human kappa lightchain constant region. The amino acid numbering for Ser 228 is accordingto the EU numbering system. See Aalberse et al., Immunology 105:9-19,2002. On day 6, cells were stained with CD8-PerCP, A0701/P7(758)-PEtetramer, PD-1-Alexa 647, CD4-Alexa 700, CD14-Alexa 700, CD16-Alexa 700,CD19-Alexa 700, and Live/Dead Blue. Samples were acquired on a BD LSR IIflow cytometer, and data was analyzed using FlowJo software.

As shown in FIG. 15, the anti-PD-L1 antibody treatment restoredHCV-specific T cell proliferation by intrahepatic mononuclear cells froma persistently infected chimpanzee.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Also incorporated by reference in their entirety are any polynucleotideand polypeptide sequences which reference an accession numbercorrelating to an entry in a public database, such as those maintainedby The Institute for Genomic Research (TIGR) on the world wide weband/or the National Center for Biotechnology Information (NCBI) on theworld wide web.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-57: (canceled) 58: A method of reactivating exhausted T cells,comprising contacting a population of T cells, wherein at least some Tcells express PD-L1, with an effective amount of a compositioncomprising an isolated antibody or an antigen-binding fragment thereof,wherein the isolated antibody, or an antigen-binding fragment thereof,comprises: a) a heavy chain variable region sequence comprising thethree CDRs with the sequences of SEQ ID NOs: 13-15; and/or b) a lightchain variable region sequence comprising the three CDRs with thesequences of SEQ ID NOs:16-18, and wherein the isolated antibody, orantigen-binding fragment thereof, binds to a PD-L1 protein having theamino acid sequence of SEQ ID NO: 4, and the isolated antibody, orantigen-binding fragment thereof, is chimeric, humanized, composite, orhuman. 59: The method of claim 58, wherein the antibody or anantigen-binding fragment thereof, comprises: a) a heavy chain variableregion sequence comprising the three CDRs with the sequences of SEQ IDNOs: 13-15; and b) a light chain variable region sequence comprising thethree CDRs with the sequences of SEQ ID NOs:16-18. 60: The method ofclaim 59, wherein the antibody or an antigen-binding fragment thereof,comprises: a) a heavy chain variable region sequence selected from thegroup consisting of SEQ ID NOs: 34-38, or a sequence with at least about95% homology to a heavy chain sequence selected from the groupconsisting of SEQ ID NOs: 34-38; and b) a light chain variable regionsequence selected from the group consisting of SEQ ID NOs: 39-42, or asequence with at least about 95% homology to a light chain sequenceselected from the group consisting of SEQ ID NOs: 39-42. 61: The methodof claim 58, wherein the antibody or an antigen-binding fragmentthereof, comprises: a) a heavy chain variable region sequence selectedfrom the group consisting of SEQ ID NOs: 34-38, or a sequence with atleast about 95% homology to a heavy chain sequence selected from thegroup consisting of SEQ ID NOs: 34-38; and/or b) a light chain variableregion sequence selected from the group consisting of SEQ ID NOs: 39-42,or a sequence with at least about 95% homology to a light chain sequenceselected from the group consisting of SEQ ID NOs: 39-42. 62: The methodof claim 61, wherein the antibody or an antigen-binding fragmentthereof, comprises: a) a heavy chain variable region sequence selectedfrom the group consisting of SEQ ID NOs: 34-38; and/or b) a light chainvariable region sequence selected from the group consisting of SEQ IDNOs: 39-42. 63: The method of claim 62, wherein the antibody or anantigen-binding fragment thereof, comprises: a) a heavy chain variableregion sequence selected from the group consisting of SEQ ID NOs: 34-38;and b) a light chain variable region sequence selected from the groupconsisting of SEQ ID NOs: 39-42. 64: The method of claim 63, wherein theantibody or an antigen-binding fragment thereof, comprises: a) a heavychain variable region sequence comprising SEQ ID NO: 35 or 37; and b) alight chain variable region sequence comprising SEQ ID NO: 39, 40 or 42.65: The method of claim 59, wherein the antibody or an antigen-bindingfragment thereof, comprises: a) a heavy chain variable region sequencecomprising SEQ ID NO: 35 or 37, or a sequence with at least about 95%homology to a heavy chain sequence comprising SEQ ID NO: 35 or 37; andb) a light chain variable region sequence comprising SEQ ID NO: 39, 40or 42, or a sequence with at least about 95% homology to a light chainsequence comprising SEQ ID NO: 39, 40 or
 42. 66: The method of claim 58,wherein the isolated antibody or antigen-binding fragment thereofinhibits the binding of an antibody comprising a heavy chain variableregion comprising the sequence of SEQ ID NO:78 and a light chainvariable region comprising the sequence of SEQ ID NO:79 to Fc-PD-L1. 67:The method of claim 58, wherein the isolated antibody or antigen-bindingfragment thereof inhibits a PD-L1-mediated signal.
 68. The method ofclaim 58, wherein the step of contacting is performed in vitro, ex vivo,or in vivo.
 69. A method of reactivating exhausted T cells, comprisingcontacting a population of T cells, wherein at least some T cellsexpress PD-1, with an effective amount of a composition comprising anisolated antibody or an antigen-binding fragment thereof, wherein theisolated antibody, or an antigen-binding fragment thereof, comprises: a)a heavy chain variable region sequence comprising the three CDRsequences of SEQ ID NOs: 7-9; and b) a light chain variable regionsequence comprising the three CDR sequences of SEQ ID NOs:10-12, andwherein the isolated antibody, or antigen-binding fragment thereof,binds to a PD-1 protein having the amino acid sequence of SEQ ID NO: 2,and the isolated antibody, or antigen-binding fragment thereof, ischimeric, humanized, composite, or human.
 70. The method of claim 69,wherein the isolated antibody or antigen-binding fragment thereofcomprises: a) a heavy chain variable region sequence selected from thegroup consisting of SEQ ID NOs: 25-29, or a sequence with at least about95% homology to a heavy chain sequence selected from the groupconsisting SEQ ID NOs: 25-29; and b) a light chain variable regionsequence selected from the group consisting of SEQ ID NOs: 30-33, or asequence with at least about 95% homology to a light chain sequenceselected from the group consisting of SEQ ID NOs: 30-33.
 71. The methodof claim 69, wherein the isolated antibody or antigen-binding fragmentthereof comprises: a) a heavy chain variable region sequence selectedfrom the group consisting of SEQ ID NOs: 25-29; and b) a light chainvariable region sequence selected from the group consisting of SEQ IDNOs: 30-33.
 72. The method of claim 71, wherein the isolated antibody orantigen-binding fragment thereof comprises: a) a heavy chain variableregion sequence comprising SEQ ID NO: 27 or 28; and b) a light chainvariable region sequence comprising SEQ ID NO: 32 or
 33. 73. The methodof claim 69, wherein the isolated antibody or antigen-binding fragmentthereof comprises: a) a heavy chain variable region sequence comprisingSEQ ID NO: 27 or 8, or a sequence with at least about 95% homology to aheavy chain sequence comprising SEQ ID NO: 27 or 28; and b) a lightchain variable region sequence comprising SEQ ID NO: 32 or 33, or asequence with at least about 95% homology to a light chain sequencecomprising SEQ ID NO: 32 or
 33. 74. The method of claim 69, wherein theisolated antibody or antigen-binding fragment thereof inhibits thebinding of an antibody comprising a heavy chain variable regioncomprising the sequence of SEQ ID NO:76 and a light chain variableregion comprising the sequence of SEQ ID NO:77 to Fc-PD-1.
 75. Themethod of claim 69, wherein the isolated antibody or antigen-bindingfragment thereof inhibits a PD-1-mediated signal.
 76. The method ofclaim 69, wherein the step of contacting is performed in vitro, ex vivo,or in vivo.